5-(Pyridin-2-yl-Amino)-Pyrazine-2-Carbonitrile Compounds and Their Therapeutic Use

ABSTRACT

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to certain pyridyl-amino-pyrazine carbonitrile compounds that, inter alia, inhibit Checkpoint Kinase 1 (CHK1) kinase function. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit CHK1 kinase function, and in the treatment of diseases and conditions that are mediated by CHK1, that are ameliorated by the inhibition of CHK1 kinase function, etc., including proliferative conditions such as cancer, etc., optionally in combination with another agent, for example, (a) a DNA topoisomerase I or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite or thymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

RELATED APPLICATION

This application is related to U.S. patent application No. 61/557,457filed 9 Nov. 2011, the contents of which are incorporated herein byreference in their entirety.

TECHNICAL FIELD

The present invention pertains generally to the field of therapeuticcompounds. More specifically the present invention pertains to certainpyridyl-amino-pyrazine carbonitrile compounds that, inter alia, inhibitCheckpoint Kinase 1 (CHK1) kinase function. The present invention alsopertains to pharmaceutical compositions comprising such compounds, andthe use of such compounds and compositions, both in vitro and in vivo,to inhibit CHK1 kinase function, and in the treatment of diseases andconditions that are mediated by CHK1, that are ameliorated by theinhibition of CHK1 kinase function, etc., including proliferativeconditions such as cancer, etc., optionally in combination with anotheragent, for example, (a) a DNA topoisomerase I or II inhibitor; (b) a DNAdamaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

BACKGROUND

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Each of these references is incorporated hereinby reference in its entirety into the present disclosure, to the sameextent as if each individual reference was specifically and individuallyindicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

Ranges are often expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by the use of the antecedent “about,” itwill be understood that the particular value forms another embodiment.

This disclosure includes information that may be useful in understandingthe present invention. It is not an admission that any of theinformation provided herein is prior art or relevant to the presentlyclaimed invention, or that any publication specifically or implicitlyreferenced is prior art.

Checkpoint Kinase 1 (CHK1)

Progression through the cell division cycle is a tightly regulatedprocess and is monitored at several positions known as cell cyclecheckpoints (see, e.g., Weinert and Hartwell, 1989; Bartek and Lukas,2003). These checkpoints are found in all four stages of the cell cycle;G1, S (DNA replication), G2 and M (Mitosis) and they ensure that keyevents which control the fidelity of DNA replication and cell divisionare completed correctly. Cell cycle checkpoints are activated by anumber of stimuli, including DNA damage and DNA errors caused bydefective replication. When this occurs, the cell cycle will arrest,allowing time for either DNA repair to occur or, if the damage is toosevere, for activation of cellular processes leading to controlled celldeath.

All cancers, by definition, have some form of aberrant cell divisioncycle. Frequently, the cancer cells possess one or more defective cellcycle checkpoints, or harbour defects in a particular DNA repairpathway. These cells are therefore often more dependent on the remainingcell cycle checkpoints and repair pathways, compared to non-cancerouscells (where all checkpoints and DNA repair pathways are intact). Theresponse of cancer cells to DNA damage is frequently a criticaldeterminant of whether they continue to proliferate or activate celldeath processes and die. For example, tumour cells that contain a mutantform(s) of the tumour suppressor p53 are defective in the G1 DNA damagecheckpoint. Thus inhibitors of the G2 or S-phase checkpoints areexpected to further impair the ability of the tumour cell to repairdamaged DNA.

Many known cancer treatments cause DNA damage by either physicallymodifying the cell's DNA or disrupting vital cellular processes that canaffect the fidelity of DNA replication and cell division, such as DNAmetabolism, DNA synthesis, DNA transcription and microtubule spindleformation. Such treatments include for example, radiotherapy, whichcauses DNA strand breaks, and a variety of chemotherapeutic agentsincluding topoisomerase inhibitors, antimetabolites, DNA-alkylatingagents, and platinum-containing cytotoxic drugs. A significantlimitation to these genotoxic treatments is drug resistance. One of themost important mechanisms leading to this resistance is attributed toactivation of cell cycle checkpoints, giving the tumour cell time torepair damaged DNA. By abrogating a particular cell cycle checkpoint, orinhibiting a particular form of DNA repair, it may therefore be possibleto circumvent tumour cell resistance to the genotoxic agents and augmenttumour cell death induced by DNA damage, thus increasing the therapeuticindex of these cancer treatments.

CHK1 is a serine/threonine kinase involved in regulating cell cyclecheckpoint signals that are activated in response to DNA damage anderrors in DNA caused by defective replication (see, e.g., Bartek andLukas, 2003). CHK1 transduces these signals through phosphorylation ofsubstrates involved in a number of cellular activities including cellcycle arrest and DNA repair. Two key substrates of CHK1 are the Cdc25Aand Cdc25C phosphatases that dephosphorylate CDK1 leading to itsactivation, which is a requirement for exit from G2 into mitosis (Mphase) (see, e.g., Sanchez et al., 1997). Phosphorylation of Cdc25C andthe related Cdc25A by CHK1 blocks their ability to activate CDK1, thuspreventing the cell from exiting G2 into M phase. The role of CHK1 inthe DNA damage-induced G2 cell cycle checkpoint has been demonstrated ina number of studies where CHK1 function has been knocked out (see, e.g.,Liu et al., 2000; Zhao et al., 2002; Zachos et al., 2003).

The reliance of the DNA damage-induced G2 checkpoint upon CHK1 providesone example of a therapeutic strategy for cancer treatment, involvingtargeted inhibition of CHK1. Upon DNA damage, the p53 tumour suppressorprotein is stabilised and activated to give a p53-dependent G1 arrest,leading to apoptosis or DNA repair (Balaint and Vousden, 2001). Overhalf of all cancers are functionally defective for p53, which can makethem resistant to genotoxic cancer treatments such as ionising radiation(IR) and certain forms of chemotherapy (see, e.g., Greenblatt et al.,1994; Carson and Lois, 1995). These p53 deficient cells fail to arrestat the G1 checkpoint or undergo apoptosis or DNA repair, andconsequently may be more reliant on the G2 checkpoint for viability andreplication fidelity. Therefore abrogation of the G2 checkpoint throughinhibition of the CHK1 kinase function may selectively sensitise p53deficient cancer cells to genotoxic cancer therapies, and this has beendemonstrated (see, e.g., Wang et al., 1996; Dixon and Norbury, 2002).

In addition, CHK1 has also been shown to be involved in S phase cellcycle checkpoints and DNA repair by homologous recombination. Thus,inhibition of CHK1 kinase in those cancers that are reliant on theseprocesses after DNA damage, may provide additional therapeuticstrategies for the treatment of cancers using CHK1 inhibitors (see,e.g., Sorensen et al., 2005). Furthermore, certain cancers may exhibitreplicative stress due to high levels of endogenous DNA damage (see,e.g., Cavalier et al., 2009; Brooks et al., 2012) or through elevatedreplication driven by oncogenes, for example amplified or overexpressedMYC genes (see, e.g., Di Micco et al. 2006; Cole et al., 2011; Murga etal. 2011). Such cancers may exhibit elevated signalling through CHK1kinase (see, e.g., Höglund et al., 2011). Inhibition of CHK1 kinase inthose cancers that are reliant on these processes, may provideadditional therapeutic strategies for the treatment of cancers usingCHK1 inhibitors (see, e.g., Cole et al., 2011; Davies et al., 2011;Ferrao et al., 2011).

Recent data using CHK1 selective siRNA supports the selective inhibitionof CHK1 as a relevant therapeutic approach, and suggests that combinedinhibition with certain other checkpoint kinases provides no additionalbenefit and may be non-productive (see, e.g., Xiao et al., 2006; Guzi etal., 2011). Small-molecule selective inhibitors of CHK1 kinase functionfrom various chemical classes have been described (see, e.g., Tao etal., 2006).

Known Compounds

Collins et al., 2009a, describes certain compounds of the followingformula which inhibit Checkpoint Kinase 1 (CHK1) kinase function, andwhich are useful in the treatment of, e.g., cancer:

Among the examples in Collins et al., 2009a are the following compounds:

Only one of the examples in Collins et al., 2009a has —R^(B6) as otherthan —H, specifically, as —OMe, while also having —X═ as —N═:

One embodiment in Collins et al., 2009a has —R^(B6) defined as“independently -Me, -Et, -nPr, -iPr, —CF₃, —OH, —OMe, —OEt, —O(nPr),—O(iPr), —OCF₃, —CN, —NH₂, —NHMe, —NMe₂, —O—CH₂CH₂—OH, —O—CH₂CH₂—OMe,—O—CH₂CH₂—NH₂, —O—CH₂CH₂—NHMe, —O—CH₂CH₂—NMe₂, —O—CH₂CH₂CH₂—NH₂,—O—CH₂CH₂CH₂—NHMe, or —O—CH₂CH₂CH₂—NMe₂” (see page 48, lines 37-40 andclaim 296 therein).

Collins et al., 2009b, describes certain compounds of the followingformula which inhibit Checkpoint Kinase 1 (CHK1) kinase function, andwhich are useful in the treatment of, e.g., cancer:

Among the examples in Collins et al., 2009b are the followingisoquinoline compounds:

Walton et al., 2010, describes preclinical studies of the CHK1 inhibitorreferred as SAR-020106, which has the following structure.

Among the examples in Collins et al., 2009b are the following1H-imidazo[4,5-b]pyridine compounds:

Almeida et al., 2008, describes certain pyrazolyl-amino-substitutedpyrazines of the following formula, which allegedly are useful in thetreatment of cancer.

Among the examples in Almeida et al., 2008 are the following compounds:

Ioannidis et al., 2009, describes certain compounds which inhibitJanus-associated kinase (JAK). The following compounds are shown inScheme 5 on page 6526 therein.

Lin et al., 2005, describes certain macrocyclic urea compounds whichallegedly are useful as protein kinase inhibitors. See, e.g., paragraph[0004] on page 1 therein.

Tao et al., 2005, describes certain macrocyclic urea compounds whichallegedly are useful as protein kinase inhibitors. See, e.g., page 2therein.

Li et al., 2007, describes the preparation and testing of certainmacrocyclic urea CHK1 inhibitors. See, e.g., Table 1 on page 6502therein.

Tao et al., 2007a, describes the preparation and testing of certainmacrocyclic urea CHK1 inhibitors. See, e.g., Table 2 on page 6596therein.

Tao et al., 2007b, describes the preparation and testing of certainmacrocyclic urea CHK1 inhibitors. See, e.g., Table 3 on page 1517therein.

One or more of the inventors have contributed to recent publications inwhich a number of CHK1 inhibitors are described, including the followingcompound, referred to as CCT244747. See, Lainchbury et al., 2012(apparently published online on 19 Oct. 2012) and Walton et al., 2012(apparently published 15 Oct. 2012).

SUMMARY OF THE INVENTION

One aspect of the invention pertains to certain pyridyl-amino-pyrazinecarbonitrile compounds (referred to herein as PAPC compounds), asdescribed herein.

Another aspect of the invention pertains to a composition (e.g., apharmaceutical composition) comprising a PAPC compound, as describedherein, and a pharmaceutically acceptable carrier or diluent.

In one embodiment, the composition (e.g., a pharmaceutical composition)is suitable for oral administration to a subject.

Another aspect of the invention pertains to a method of preparing acomposition (e.g., a pharmaceutical composition) comprising the step ofadmixing a PAPC compound, as described herein, and a pharmaceuticallyacceptable carrier or diluent.

Another aspect of the present invention pertains to a method ofinhibiting CHK1 kinase function in a cell, in vitro or in vivo,comprising contacting the cell with an effective amount of a PAPCcompound, as described herein.

In one embodiment, the method further comprises contacting the cell withone or more other agents selected from: (a) a DNA topoisomerase I or IIinhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Another aspect of the present invention pertains to a method ofregulating (e.g., inhibiting) cell proliferation (e.g., proliferation ofa cell), inhibiting cell cycle progression, promoting cell apoptosis, ora combination of one or more these, in vitro or in vivo, comprisingcontacting a cell with an effective amount of a PAPC compound, asdescribed herein.

In one embodiment, the method further comprises contacting the cell withone or more other agents selected from: (a) a DNA topoisomerase I or IIinhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Another aspect of the present invention pertains to a method oftreatment comprising administering to a subject in need of treatment atherapeutically-effective amount of a PAPC compound, as describedherein, preferably in the form of a pharmaceutical composition.

In one embodiment, said administering is orally administering.

In one embodiment, the method further comprises administering to thesubject one or more other agents selected from: (a) a DNA topoisomeraseI or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Another aspect of the present invention pertains to a PAPC compound asdescribed herein for use in a method of treatment of the human or animalbody by therapy.

In one embodiment, the compound is for use in a method of treatment ofthe human or animal body by therapy by oral administration.

In one embodiment, the method of treatment comprises treatment with both(i) a PAPC compound and (ii) one or more other agents selected from: (a)a DNA topoisomerase I or II inhibitor; (b) a DNA damaging agent; (c) anantimetabolite or thymidylate synthase (TS) inhibitor; (d) a microtubuletargeted agent; and (e) ionising radiation.

Another aspect of the present invention pertains to use of a PAPCcompound, as described herein, in the manufacture of a medicament foruse in treatment.

In one embodiment, the medicament is a medicament for oraladministration.

In one embodiment, the treatment comprises treatment with both (i) amedicament comprising a PAPC compound and (ii) one or more other agentsselected from: (a) a DNA topoisomerase I or II inhibitor; (b) a DNAdamaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

In one embodiment, the treatment is treatment of a disease or conditionthat is mediated by CHK1.

In one embodiment, the treatment is treatment of a disease or conditionthat is ameliorated by the inhibition of CHK1 kinase function.

In one embodiment, the treatment is treatment of a proliferativecondition.

In one embodiment, the treatment is treatment of cancer.

In one embodiment, the treatment is treatment of p53 deficient cancer.

In one embodiment, the treatment is treatment of head cancer; neckcancer; nervous system cancer; brain cancer; neuroblastoma;lung/mediastinum cancer; breast cancer; oesophagus cancer; stomachcancer; liver cancer; biliary tract cancer; pancreatic cancer; smallbowel cancer; large bowel cancer; colorectal cancer; gynaecologicalcancer; genito-urinary cancer; ovarian cancer; thyroid gland cancer;adrenal gland cancer; skin cancer; melanoma; bone sarcoma; soft tissuesarcoma; paediatric malignancy; Hodkin's disease; non-Hodgkin'slymphoma; myeloma; leukaemia; or metastasis from an unknown primarysite.

In one embodiment, the treatment is treatment of: lung cancer, breastcancer, ovarian cancer, colorectal cancer, melanoma, glioma, orneuroblastoma.

Another aspect of the present invention pertains to a kit comprising (a)a PAPC compound, as described herein, preferably provided as apharmaceutical composition and in a suitable container and/or withsuitable packaging; and (b) instructions for use, for example, writteninstructions on how to administer the compound.

In one embodiment, the kit further comprises one or more other agentsselected from: (a) a DNA topoisomerase I or II inhibitor; (b) a DNAdamaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; and (d) a microtubule targeted agent.

Another aspect of the present invention pertains to a PAPC compoundobtainable by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to a PAPC compoundobtained by a method of synthesis as described herein, or a methodcomprising a method of synthesis as described herein.

Another aspect of the present invention pertains to novel intermediates,as described herein, which are suitable for use in the methods ofsynthesis described herein.

Another aspect of the present invention pertains to the use of suchnovel intermediates, as described herein, in the methods of synthesisdescribed herein.

As will be appreciated by one of skill in the art, features andpreferred embodiments of one aspect of the invention will also pertainto other aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an MRI scan recorded as part of the in vivo study (“PAPC-A-01in Transgenic MYCN-driven Neuroblastoma Model”) described below. Theimage shows the abdomen of a mouse (k=kidney; t=tumour; s.b.=smallbowel) and was recorded pre-treatment. The tumour volume was 1960 mm³.

FIG. 2 is an MRI scan recorded as part of the in vivo study (“PAPC-A-01in Transgenic MYCN-driven Neuroblastoma Model”) described below. Theimage shows the abdomen of a mouse (k=kidney; t=tumour; s.b.=smallbowel) and was recorded following 7 days treatment with PAPC-A-01. Thetumour volume was 417 mm³.

DETAILED DESCRIPTION OF THE INVENTION Compounds

One aspect of the present invention relates to certain compounds thatare related to 5-(pyridin-2-yl-amino)-pyrazine-2-carbonitrile:

All of the compounds are additionally characterised by a substituentadjacent to the carbonitrile group (at the 3-position of the pyrazine)that is independently:

wherein R^(B3A) is as defined herein. These two formulae define groupswhich may conveniently be described as “open chain” (on the left) and“closed ring” (on the right) analogues of each other, and share theatoms/bonds marked in bold below:

For example, when R^(B3A) is methyl, the compounds are related to thefollowing compounds:

The compounds of the present invention are potent inhibitors of CHK1activity (e.g., having a CHK1 IC₅₀ of less than 100 nM). The compoundsof the present invention may additionally be characterised by (a)notable selectivity as compared to CHK2 (e.g., a CHK1 vs. CHK2selectivity of at least 100-fold) and/or (b) notable oralbioavailability (e.g., oral bioavailability of at least 100 nM (plasmaconcentration, 1 hour following 10 mg/kg p.o.)).

Thus, one aspect of the present invention pertains to compounds of thefollowing formula, and pharmaceutically acceptable salts, hydrates, andsolvates thereof, wherein —R^(A4)—R^(A5), and —R^(B3) are as definedherein (for convenience, collectively referred to herein as“pyridyl-amino-pyrazine carbonitrile compounds” or “PAPC compounds”):

Some embodiments of the invention include the following:

(1) A compound of the following formula, or a pharmaceuticallyacceptable salt, hydrate, or solvate thereof:

wherein:

-   -   —R^(B3) is independently:

-   -   each —R^(B3A) is independently —H or saturated aliphatic        C₁₋₃alkyl;    -   —R^(A4) is independently —NHR^(A4A), —NR^(A4A) ₂, or —OR^(A4A);    -   each —R^(MA) is independently saturated aliphatic C₁₋₃alkyl;    -   —R^(A5) is independently —R^(A5A), —R^(A5B), —R^(A5C), —R^(A5D),        —R^(A5E), or —R^(A5F);    -   —R^(A5A) is independently:

-   -   —R^(A5AA) is saturated aliphatic C₁₋₃alkyl;    -   —R^(A5B) is —CF₃;    -   R^(A5C) is independently —F, —Cl, —Br, or —I;    -   —R^(A5D) is independently —C≡CH, —C≡C—R^(A5DA), or        —C≡C—R^(A5DB)—OH;    -   —R^(A5DA) is saturated aliphatic C₁₋₄alkyl;

—R^(A5DB) is saturated aliphatic C₁₋₄alkylene;

-   -   —R^(A5E) is independently saturated C₃₋₆cycloalkyl;    -   —R^(A5F) is —C(═O)O—R^(A5FA); and    -   —R^(A5FA) is saturated aliphatic C₁₋₃alkyl.

For the avoidance of doubt, it is not intended that any two or more of—R^(A4), —R^(A5), and —R^(B3) together form a ring fused to the ring(s)to which they are attached. For example, it is not intended that —R^(A4)and —R^(A5) together form a ring fused to the ring to which they areattached. Similarly, it is not intended that —R^(A4) and —R^(B3)together form a ring fused to the rings to which they are attached.Similarly, it is not intended that —R^(A5) and —R^(B3) together form aring fused to the rings to which they are attached.

The group —R^(B3) has one chiral centre, marked by an asterisk in thefollowing formulae, which may independently be in the (R) or (S)configuration. Unless otherwise indicated, it is intended that bothconfigurations are encompassed.

The Group —R^(B3)

(2) A compound according to (1), wherein —R^(B3) is:

(3) A compound according to (1), wherein —R^(B3) is:

(4) A compound according to (1), wherein —R^(B3) is:

(5) A compound according to (1), wherein —R^(B3) is:

(6) A compound according to (1), wherein —R^(B3) is:

(7) A compound according to (1), wherein —R^(B3) is:

The Group —R^(B3A)

(8) A compound according to any one of (1) to (7), wherein —R^(B3A) issaturated aliphatic C₁₋₃alkyl.

(9) A compound according to any one of (1) to (7), wherein —R^(B3A) is-Me.

(10) A compound according to any one of (1) to (7), wherein —R^(B3A) is—H.

The Group —R^(A4)

(11) A compound according to any one of (1) to (10), wherein —R^(A4) isindependently —NHR^(A4A) or —NR^(A4A) ₂.

(12) A compound according to any one of (1) to (10), wherein —^(RA4) is—NHR^(A4A).

(13) A compound according to any one of (1) to (10), wherein —R^(A4) is—NR^(A4A) ₂.

(14) A compound according to any one of (1) to (10), wherein —R^(A4) is—OR^(A4A).

The Group —R^(A4A)

(15) A compound according to any one of (1) to (14), wherein each—R^(A4A) is independently -Me or -Et.

(16) A compound according to any one of (1) to (14), wherein each—R^(A4A) is -Me.

The Group —R^(A5)

(17) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5A).

(18) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5B).

(19) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5C).

(20) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5D).

(21) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5E).

(22) A compound according to any one of (1) to (16), wherein —R^(A5) is—R^(A5F).

The Group —R^(A5A)

(23) A compound according to any one of (1) to (22), wherein —R^(A5A),if present, is:

(24) A compound according to any one of (1) to (22), wherein —R^(A5A),if present, is:

The Group —R^(A5AA)

(25) A compound according to any one of (1) to (24), wherein each—R^(A5AA), if present, is -Me or -Et.

(26) A compound according to any one of (1) to (24), wherein each—R^(A5AA), if present, is -Me.

(27) A compound according to any one of (1) to (24), wherein each—R^(A5AA), if present, is -Et.

The Group —R^(A5C)

(28) A compound according to any one of (1) to (27), wherein —R^(A5C),if present, is independently —F, —Cl, or —Br.

(29) A compound according to any one of (1) to (27), wherein —R^(A5C),if present, is independently —F.

(30) A compound according to any one of (1) to (27), wherein —R^(A5C),if present, is independently —Cl.

(31) A compound according to any one of (1) to (27), wherein —R^(A5C),if present, is independently —Br.

(32) A compound according to any one of (1) to (27), wherein —R^(A5C),if present, is independently —I.

The Group —R^(A5D)

(33) A compound according to any one of (1) to (32), wherein —R^(A5D),if present, is independently —C≡CH or —C≡C—R^(A5DA).

(34) A compound according to any one of (1) to (32), wherein —R^(A5D),if present, is —C≡CH.

(35) A compound according to any one of (1) to (32), wherein —R^(A5D),if present, is —C≡C—R^(A5DA).

(36) A compound according to any one of (1) to (32), wherein —R^(A5D),if present, is —C≡C—R^(A5DB)—OH.

The Group —R^(A5DA)

(37) A compound according to any one of (1) to (36), wherein —R^(A5DA),if present, is independently -Me, -Et, —CH(Me)₂, or —C(Me)₃.

(38) A compound according to any one of (1) to (36), wherein —R^(A5DA),if present, is —CH(Me)₂.

(39) A compound according to any one of (1) to (36), wherein —R^(A5DA),if present, is —C(Me)₃.

The Group —R^(A5DB)—

(40) A compound according to any one of (1) to (39), wherein —R^(A5DB)—,if present, is saturated aliphatic C₁₋₃alkylene.

(41) A compound according to any one of (1) to (39), wherein —R^(A5DB)—,if present, is independently —CH₂—, —CH(Me)—, or —C(Me)₂-.

(42) A compound according to any one of (1) to (39), wherein —R^(A5DB)—,if present, is —C(Me)₂-.

(43) A compound according to any one of (1) to (39), wherein —R^(A5DB)—,if present, is —CH(Me)-.

(44) A compound according to any one of (1) to (39), wherein —R^(A5DB)—,if present, is —CH₂—.

The Group —R^(A5E)

(45) A compound according to any one of (1) to (44), wherein —R^(A5E),if present, is independently cyclopropyl, cyclobutyl, or cyclopentyl.

(46) A compound according to any one of (1) to (44), wherein —R^(A5E),if present, is independently cyclopropyl or cyclobutyl.

(47) A compound according to any one of (1) to (44), wherein —R^(A5E),if present, is cyclopropyl.

The Group —R^(A5FA)

(48) A compound according to any one of (1) to (47), wherein —R^(A5FA),if present, is -Me or -Et.

(49) A compound according to any one of (1) to (47), wherein —R^(A5FA),if present, is -Me.

(50) A compound according to any one of (1) to (47), wherein —R^(A5FA),if present, is -Et.

Some Preferred Combinations

(51) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(52) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(53) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(54) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(55) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(56) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(57) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(58) A compound according to (1), which is a compound of the followingformula, or a pharmaceutically acceptable salt, hydrate, or solvatethereof:

(59) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(60) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(61) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(62) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(63) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(64) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(65) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(66) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(67) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

(68) A compound according to (1), which is a compound of one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

Specific Compounds

(69) A compound according to (1), which is a compound one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

Code No. Structure PAPC- A-01

PAPC- A-02

PAPC- A-03

PAPC- A-04

PAPC- A-05

PAPC- A-06

PAPC- A-07

PAPC- A-08

PAPC- A-09

PAPC- A-10

(70) A compound according to (1), which is a compound one of thefollowing formulae, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof:

Code No. Structure PAPC- B-01

PAPC- B-02

PAPC- B-03

PAPC- B-04

PAPC- B-05

PAPC- B-06

PAPC- B-07

PAPC- B-08

PAPC- B-09

PAPC- B-10

PAPC- B-11

Combinations

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables (e.g.,—R^(B3), —R^(B3A), —R^(A4), —R^(A4A), —R^(A5), —R^(A5A), —R^(A5AA),—R^(A5B), —R^(A5C), —R^(A5D), —R^(A5DA), —R^(A5DB)—, —R^(A5E), —R^(A5F),—R^(A5FA), etc.) are specifically embraced by the present invention andare disclosed herein just as if each and every combination wasindividually and explicitly disclosed, to the extent that suchcombinations embrace compounds that are stable compounds (i.e.,compounds that can be isolated, characterised, and tested for biologicalactivity). In addition, all sub-combinations of the chemical groupslisted in the embodiments describing such variables are alsospecifically embraced by the present invention and are disclosed hereinjust as if each and every such sub-combination of chemical groups wasindividually and explicitly disclosed herein.

Substantially Purified Forms

One aspect of the present invention pertains to PAPC compounds, inpurified form.

In one embodiment, the compound is in substantially purified form and/orin a form substantially free from contaminants.

In one embodiment, the compound is in a substantially purified form witha purity of least 50% by weight, e.g., at least 60% by weight, e.g., atleast 70% by weight, e.g., at least 80% by weight, e.g., at least 90% byweight, e.g., at least 95% by weight, e.g., at least 97% by weight,e.g., at least 98% by weight, e.g., at least 99% by weight.

Unless specified, the substantially purified form refers to the compoundin any stereoisomeric or enantiomeric form. For example, in oneembodiment, the substantially purified form refers to a mixture ofstereoisomers, i.e., purified with respect to other compounds. In oneembodiment, the substantially purified form refers to one stereoisomer,e.g., optically pure stereoisomer. In one embodiment, the substantiallypurified form refers to a mixture of enantiomers. In one embodiment, thesubstantially purified form refers to an equimolar mixture ofenantiomers (i.e., a racemic mixture, a racemate). In one embodiment,the substantially purified form refers to one enantiomer, e.g.,optically pure enantiomer.

In one embodiment, the compound is in a form substantially free fromcontaminants wherein the contaminants represent no more than 50% byweight, e.g., no more than 40% by weight, e.g., no more than 30% byweight, e.g., no more than 20% by weight, e.g., no more than 10% byweight, e.g., no more than 5% by weight, e.g., no more than 3% byweight, e.g., no more than 2% by weight, e.g., no more than 1% byweight.

Unless specified, the contaminants refer to other compounds, that is,other than stereoisomers or enantiomers. In one embodiment, thecontaminants refer to other compounds and other stereoisomers. In oneembodiment, the contaminants refer to other compounds and the otherenantiomer.

In one embodiment, the compound is in a substantially purified form withan optical purity of at least 60% (i.e., 60% of the compound, on a molarbasis, is the desired stereoisomer or enantiomer, and 40% is undesiredstereoisomer(s) or enantiomer), e.g., at least 70%, e.g., at least 80%,e.g., at least 90%, e.g., at least 95%, e.g., at least 97%, e.g., atleast 98%, e.g., at least 99%.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g., C₁₋₃alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including mixtures (e.g., racemicmixtures) thereof. Methods for the preparation (e.g., asymmetricsynthesis) and separation (e.g., fractional crystallisation andchromatographic means) of such isomeric forms are either known in theart or are readily obtained by adapting the methods taught herein, orknown methods, in a known manner.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, formic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Hydrates and Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the compound. The term “solvate” is used hereinin the conventional sense to refer to a complex of solute (e.g.,compound, salt of compound) and solvent. If the solvent is water, thesolvate may be conveniently referred to as a hydrate, for example, ahemi-hydrate, a mono-hydrate, a sesqui-hydrate, a di-hydrate, atri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound alsoincludes solvate and hydrate forms thereof.

Chemically Protected Forms

It may be convenient or desirable to prepare, purify, and/or handle thecompound in a chemically protected form. The term “chemically protectedform” is used herein in the conventional chemical sense and pertains toa compound in which one or more reactive functional groups are protectedfrom undesirable chemical reactions under specified conditions (e.g.,pH, temperature, radiation, solvent, and the like). In practice, wellknown chemical methods are employed to reversibly render unreactive afunctional group, which otherwise would be reactive, under specifiedconditions. In a chemically protected form, one or more reactivefunctional groups are in the form of a protected or protecting group(also known as a masked or masking group or a blocked or blockinggroup). By protecting a reactive functional group, reactions involvingother unprotected reactive functional groups can be performed, withoutaffecting the protected group; the protecting group may be removed,usually in a subsequent step, without substantially affecting theremainder of the molecule. See, for example, Protective Groups inOrganic Synthesis (T. Greene and P. Wuts; 4th Edition; John Wiley andSons, 2006).

A wide variety of such “protecting,” “blocking,” or “masking” methodsare widely used and well known in organic synthesis. For example, acompound which has two nonequivalent reactive functional groups, both ofwhich would be reactive under specified conditions, may be derivatizedto render one of the functional groups “protected,” and thereforeunreactive, under the specified conditions; so protected, the compoundmay be used as a reactant which has effectively only one reactivefunctional group. After the desired reaction (involving the otherfunctional group) is complete, the protected group may be “deprotected”to return it to its original functionality.

For example, a hydroxy group may be protected as an ether (—OR) or anester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl,benzhydryl(diphenylmethyl), or trityl(triphenylmethyl)ether; atrimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester(—OC(═O)CH₃, —OAc).

For example, an amine group may be protected, for example, as an amide(—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide(—NHCO—CH₃); a benzyloxy amide (—NHCO—OCH₂C₆H₅, —NH-Cbz); as a t-butoxyamide (—NHCO—OC(CH₃)₃, —NH-Boc); a 2-biphenyl-2-propoxy amide(—NHCO—OC(CH₃)₂C₆H₄C₆H₅, —NH-Bpoc), as a 9-fluorenylmethoxy amide(—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxyamide (—NH-Troc), as an allyloxy amide (—NH-Alloc), as a2(-phenylsulfonyl)ethyloxy amide (—NH-Psec); or, in suitable cases(e.g., cyclic amines), as a nitroxide radical (>N—O.).

Prodrugs

It may be convenient or desirable to prepare, purify, and/or handle thecompound in the form of a prodrug. The term “prodrug,” as used herein,pertains to a compound which, when metabolised (e.g., in vivo), yieldsthe desired active compound. Typically, the prodrug is inactive, or lessactive than the desired active compound, but may provide advantageoushandling, administration, or metabolic properties.

For example, some prodrugs are esters of the active compound (e.g., aphysiologically acceptable metabolically labile ester). Duringmetabolism, the ester group (—C(═O)OR) is cleaved to yield the activedrug. Such esters may be formed by esterification, for example, of anyof the carboxylic acid groups (—C(═O)OH) in the parent compound, with,where appropriate, prior protection of any other reactive groups presentin the parent compound, followed by deprotection if required.

Also, some prodrugs are activated enzymatically to yield the activecompound, or a compound which, upon further chemical reaction, yieldsthe active compound (for example, as in ADEPT, GDEPT, LIDEPT, etc.). Forexample, the prodrug may be a sugar derivative or other glycosideconjugate, or may be an amino acid ester derivative.

General Chemical Synthesis

Several methods for the chemical synthesis of PAPC compounds aredescribed herein. These and/or other well known methods may be modifiedand/or adapted in known ways in order to facilitate the synthesis ofadditional compounds described herein.

Chemical Synthesis

Several methods for the chemical synthesis of pyridyl-amino-pyrazinecarbonitrile (PAPC) compounds of the present invention are describedherein. These and/or other well known methods may be modified and/oradapted in known ways in order to facilitate the synthesis of additionalcompounds within the scope of the present invention.

In one approach (General Method A), compounds of type (v) are preparedby a method as illustrated in the following scheme. Commerciallyavailable compound (i) is reacted with a source of ammonia, typically inaqueous solution with heating, to give aminopyrazine (ii). Subsequentreaction with a brominating agent, such as N-bromosuccinimide at 0° C.,gives the bromopyrazine (iii). Subsequent reaction of the bromopyrazinewith a cyanide source, typically potassium cyanide, under palladiummediated coupling conditions, gives the pyrazine carbonitrile (iv).Subsequent reaction with an alcohol, typically in an aprotic solventsuch as dioxane in the presence of a base such as sodium hydride,typically with heating, gives the required6-alkoxy-substituted-2-aminopyrazine-5-carbonitriles (v).

In another approach (General Method B), compounds of type (viii) areprepared by a method as illustrated in the following scheme.Commercially available compound (vi) is reacted with an aldehyde (vii),for example paraformaldehyde, under reductive amination conditions usinga reducing agent, for example sodium triacetoxyborohydride, typicallywith heating and in the presence of an acid, to give the requiredcompounds (viii).

In another approach (General Method C), compounds of type (x) areprepared by a method as illustrated in the following scheme. Compounds(ix) are reacted with an iodinating agent, typically N-iodosuccinimidein sulphuric acid, to afford the required compounds (x).

In another approach (General Method D), compounds of type (xii) areprepared by a method as illustrated in the following scheme. Compounds(xi) are reacted with a chlorinating agent, typicallyN-chlorosuccinimide in acetic acid, to afford the required compounds(xii).

In another approach (General Method E), compounds of type (xv) areprepared by a method as illustrated in the following scheme.Commercially available compound (xiii) is reacted with an amine (xiv),for example methylamine, typically with heating in a microwave reactor,to afford the required compounds (xv).

In another approach (General Method F), compounds of type (xvii) areprepared by a method as illustrated in the following scheme. Compounds(xvi) are reacted with an amine (xiv), for example dimethylamine,typically in acetonitrile at room temperature or below, to afford therequired compounds (xvii).

In another approach (General Method G), compounds of type (xix) areprepared by a method as illustrated in the following scheme. Compounds(xvi) are reacted with an alkoxide salt (xviii), for example sodiummethoxide, in an aprotic solvent such as tetrahydrofuran at roomtemperature, to afford the required compounds (xix).

In another approach (General Method H), compounds of type (xxii) areprepared by a method as illustrated in the following scheme. Compounds(xx) are reacted with haloalkanes (xxi), for example iodomethane,typically in an aprotic solvent such as DMF and in the presence of abase, such as sodium hydride, to afford the required compounds (xxii).

In another approach (General Method I), compounds of type (xxv) areprepared by a method as illustrated in the following scheme.5-Iodo-2-chloropyridines (viii) or (x) are coupled with boronic acids oresters (xxiii), for example 1-methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole or2-cyclopropyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, under palladiummediated coupling conditions in a solvent such as acetonitrile,typically with oil bath or microwave heating, and typically in thepresence of a metal carbonate base, to provide pyridines (xxiv).Treatment of intermediates (xxiv) with pyrazine compounds (v) underpalladium mediated amination conditions, typically with microwave or oilbath heating and in the presence of a base, such as a metal carbonate,gives, after removal of any protecting groups, the required PAPCcompounds (xxv).

In another approach (General Method J), compounds of type (xxvi) areprepared by a method as illustrated in the following scheme.2-Chloropyridine-5-carboxylate esters (xvii) or (xix), are coupled withpyrazine compounds (v) under palladium mediated amination conditions,typically with microwave or oil bath heating and in the presence of abase, such as a metal carbonate, to give, after removal of anyprotecting groups, the required PAPC compounds (xxvi).

In another approach (General Method K), compounds of type (xxvii) areprepared by a method as illustrated in the following scheme.2,5-Dichloropyridines (xii) are coupled with pyrazine compounds (v)under palladium mediated amination conditions, typically with microwaveor oil bath heating and in the presence of a base, such as a metalcarbonate, to give, after removal of any protecting groups, the requiredPAPC compounds (xxvii).

In another approach (General Method L), compounds of type (xxviii) areprepared by a method as illustrated in the following scheme.5-Trifluoromethyl-2-chloropyridines (xv) are coupled with pyrazinecompounds (v) under palladium mediated amination conditions, typicallywith microwave or oil bath heating and in the presence of a base, suchas a metal carbonate, to give, after removal of any protecting groups,the required PAPC compounds (xxviii).

In another approach (General Method M), compounds of type (xxxi) areprepared by a method as illustrated in the following scheme.5-Iodo-2-chloropyridines (viii) or (x) are coupled with alkynes (xxix),for example ethynyltrimethylsilane ortrimethyl(2-methylbut-3-yn-2-yloxy)silane under palladium mediatedcoupling conditions in the presence of a copper (I) salt, for examplecopper (I) iodide, in a solvent such as DMF typically with oil bath ormicrowave heating, and typically in the presence of a base, to providepyridines (xxx). Treatment of intermediates (xxx) with pyrazinecompounds (v) under palladium mediated amination conditions, typicallywith microwave or oil bath heating and in the presence of a base, suchas a metal carbonate, gives, after removal of any protecting groups, therequired PAPC compounds (xxxi).

In another approach (General Method N), compounds of type (xxxii) areprepared by a method as illustrated in the following scheme.5-(Pyrazol-4-yl)-2-chloropyridines (xxii), are coupled with pyrazinecompounds (v) under palladium mediated amination conditions, typicallywith microwave or oil bath heating and in the presence of a base, suchas a metal carbonate, to give, after removal of any protecting groups,the required PAPC compounds (xxxii).

Compositions

One aspect of the present invention pertains to a composition (e.g., apharmaceutical composition) comprising a PAPC compound, as describedherein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

Another aspect of the present invention pertains to a method ofpreparing a composition (e.g., a pharmaceutical composition) comprisingadmixing a PAPC compound, as described herein, and a pharmaceuticallyacceptable carrier, diluent, or excipient.

In one preferred embodiment, the composition (e.g., a pharmaceuticalcomposition) is suitable for oral administration to a subject.

Uses

The PAPC compounds, as described herein, are useful, for example, in thetreatment of disorders (e.g., diseases) that are ameliorated by theinhibition of CHK1 kinase function, as described herein.

Use in Methods of Inhibiting CHK1

One aspect of the present invention pertains to a method of inhibitingCHK1 kinase function, in vitro or in vivo, comprising contacting a CHK1kinase with an effective amount of a PAPC compound, as described herein.

One aspect of the present invention pertains to a method of inhibitingCHK1 kinase function in a cell, in vitro or in vivo, comprisingcontacting the cell with an effective amount of a PAPC compound, asdescribed herein.

In one embodiment, the method further comprises contacting the cell withone or more other agents selected from: (a) a DNA topoisomerase I or IIinhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Suitable assays for determining CHK1 kinase function inhibition aredescribed herein and/or are known in the art.

In one embodiment, the method is performed in vitro.

In one embodiment, the method is performed in vivo.

In one embodiment, the PAPC compound is provided in the form of apharmaceutically acceptable composition.

Any type of cell may be treated, including but not limited to, adipose,lung, gastrointestinal (including, e.g., bowel, colon), breast(mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder,pancreas, brain, and skin.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound inhibits CHK1 kinase function. For example,suitable assays are described herein.

Use in Methods of Inhibiting Cell Proliferation, Etc.

The PAPC compounds described herein, e.g., (a) regulate (e.g., inhibit)cell proliferation; (b) inhibit cell cycle progression; (c) promote cellapoptosis; or (d) a combination of one or more of these.

One aspect of the present invention pertains to a method of regulating(e.g., inhibiting) cell proliferation (e.g., proliferation of a cell),inhibiting cell cycle progression, promoting cell apoptosis, or acombination of one or more these, in vitro or in vivo, comprisingcontacting a cell with an effective amount of a PAPC compound, asdescribed herein.

In one embodiment, the method is a method of regulating (e.g.,inhibiting) cell proliferation (e.g., proliferation of a cell), in vitroor in vivo, comprising contacting a cell with an effective amount of aPAPC compound, as described herein.

In one embodiment, the method further comprises contacting the cell withone or more other agents selected from: (a) a DNA topoisomerase I or IIinhibitor; (b) a DNA damaging agent; (c) an antimetabolite or TSinhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

In one embodiment, the method is performed in vitro.

In one embodiment, the method is performed in vivo.

In one embodiment, the PAPC compound is provided in the form of apharmaceutically acceptable composition.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g., bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound regulates (e.g., inhibits) cell proliferation,etc. For example, assays which may conveniently be used to assess theactivity offered by a particular compound are described herein.

For example, a sample of cells (e.g., from a tumour) may be grown invitro and a compound brought into contact with said cells, and theeffect of the compound on those cells observed. As an example of“effect,” the morphological status of the cells (e.g., alive or dead,etc.) may be determined. Where the compound is found to exert aninfluence on the cells, this may be used as a prognostic or diagnosticmarker of the efficacy of the compound in methods of treating a patientcarrying cells of the same cellular type.

Use in Methods of Therapy

Another aspect of the present invention pertains to a PAPC compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy.

Another aspect of the present invention pertains to a PAPC compound, asdescribed herein, for use in a method of treatment of the human oranimal body by therapy by oral administration.

In one embodiment, the method of treatment comprises treatment with both(i) a PAPC compound, as described herein, and (ii) one or more otheragents selected from: (a) a DNA topoisomerase I or II inhibitor; (b) aDNA damaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

Another aspect of the present invention pertains to (a) a DNAtopoisomerase I or II inhibitor, (b) a DNA damaging agent, (c) anantimetabolite or thymidylate synthase (TS) inhibitor, or (d) amicrotubule targeted agent, as described herein, for use in a method oftreatment of the human or animal body by therapy, wherein the method oftreatment comprises treatment with both (i) a PAPC compound, asdescribed herein, and (a) the DNA topoisomerase I or II inhibitor, (b)the DNA damaging agent, (c) the antimetabolite or thymidylate synthase(TS) inhibitor, or (d) the microtubule targeted agent.

Use in the Manufacture of Medicaments

Another aspect of the present invention pertains to use of a PAPCcompound, as described herein, in the manufacture of a medicament foruse in treatment.

Another aspect of the present invention pertains to use of a PAPCcompound, as described herein, in the manufacture of a medicament foruse in treatment by oral administration.

In one embodiment, the medicament comprises the PAPC compound.

In one embodiment, the treatment comprises treatment with both (i) amedicament comprising a PAPC compound, as described herein, and (ii) oneor more other agents selected from: (a) a DNA topoisomerase I or IIinhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Another aspect of the present invention pertains to use of (a) a DNAtopoisomerase I or II inhibitor, (b) a DNA damaging agent, (c) anantimetabolite or TS inhibitor, or (d) a microtubule targeted agent, asdescribed herein, in the manufacture of a medicament for use in atreatment, wherein the treatment comprises treatment with both (i) aPAPC compound, as described herein, and (a) the DNA topoisomerase I orII inhibitor, (b) the DNA damaging agent, (c) the antimetabolite orthymidylate synthase (TS) inhibitor, or (d) the microtubule targetedagent.

Methods of Treatment

Another aspect of the present invention pertains to a method oftreatment comprising administering to a patient in need of treatment atherapeutically effective amount of a PAPC compound, as describedherein, preferably in the form of a pharmaceutical composition.

Another aspect of the present invention pertains to a method oftreatment comprising orally administering to a patient in need oftreatment a therapeutically effective amount of a PAPC compound, asdescribed herein, preferably in the form of a pharmaceuticalcomposition.

In one embodiment, the method further comprises administering to thesubject one or more other agents selected from: (a) a DNA topoisomeraseI or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite orthymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent;and (e) ionising radiation.

Conditions Treated—Conditions Mediated by CHK1

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of a disease or condition that is mediated by CHK1.

Conditions Treated—Conditions Ameliorated by the Inhibition of CHK1Kinase Function

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of: a disease or condition that is ameliorated by theinhibition of CHK1 kinase function.

Disorders Treated—Proliferative Conditions

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of: a proliferative condition.

The term “proliferative condition,” as used herein, pertains to anunwanted or uncontrolled cellular proliferation of excessive or abnormalcells which is undesired, such as neoplastic or hyperplastic growth.

In one embodiment, the treatment is treatment of: a proliferativecondition characterised by benign, pre-malignant, or malignant cellularproliferation, including for example: neoplasms, hyperplasias, andtumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (seebelow), psoriasis, bone diseases, fibroproliferative disorders (e.g., ofconnective tissues), pulmonary fibrosis, atherosclerosis, smooth musclecell proliferation in the blood vessels, such as stenosis or restenosisfollowing angioplasty.

Disorders Treated—Cancer

In one embodiment (e.g., of use in methods of therapy, of use in themanufacture of medicaments, of methods of treatment), the treatment istreatment of cancer.

In one embodiment, the treatment is treatment of lung cancer, small celllung cancer, non-small cell lung cancer, gastrointestinal cancer,stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectalcancer, thyroid cancer, breast cancer, ovarian cancer, endometrialcancer, prostate cancer, testicular cancer, liver cancer, kidney cancer,renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer,neuroblastoma, glioma, sarcoma, osteosarcoma, bone cancer,nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer,squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma,lymphoma, or leukemia.

In one embodiment, the treatment is treatment of:

-   -   a carcinoma, for example a carcinoma of the bladder, breast,        colon (e.g., colorectal carcinomas such as colon adenocarcinoma        and colon adenoma), kidney, epidermal, liver, lung (e.g.,        adenocarcinoma, small cell lung cancer and non-small cell lung        carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g.,        exocrine pancreatic carcinoma), stomach, cervix, thyroid,        prostate, skin (e.g., squamous cell carcinoma);    -   a hematopoietic tumour of lymphoid lineage, for example        leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell        lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell        lymphoma, or Burkett's lymphoma;    -   a hematopoietic tumor of myeloid lineage, for example acute and        chronic myelogenous leukemias, myelodysplastic syndrome, or        promyelocytic leukemia;    -   a tumour of mesenchymal origin, for example fibrosarcoma or        habdomyosarcoma;    -   a tumor of the central or peripheral nervous system, for example        astrocytoma, neuroblastoma, glioma or schwannoma;    -   melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma        pigmentoum; keratoctanthoma; thyroid follicular cancer; or        Kaposi's sarcoma.

In one embodiment, the treatment is treatment of solid tumour cancer.

In one embodiment, the treatment is treatment of head cancer; neckcancer; nervous system cancer; brain cancer; neuroblastoma;lung/mediastinum cancer; breast cancer; oesophagus cancer; stomachcancer; liver cancer; biliary tract cancer; pancreatic cancer; smallbowel cancer; large bowel cancer; colorectal cancer; gynaecologicalcancer; genito-urinary cancer; ovarian cancer; thyroid gland cancer;adrenal gland cancer; skin cancer; melanoma; bone sarcoma; soft tissuesarcoma; paediatric malignancy; Hodgkin's disease; non-Hodgkin'slymphoma; myeloma; leukaemia; or metastasis from an unknown primarysite.

In one embodiment, the cancer is characterised by, or furthercharacterised by, being p53 deficient cancer. In one embodiment, thecancer is p53 deficient cancer.

In one embodiment, the treatment is treatment of cancer metastasis.

In one embodiment, the cancer is characterised by, or furthercharacterised by, cancer stem cells.

The anti-cancer effect may arise through one or more mechanisms,including but not limited to, the regulation of cell proliferation, theinhibition of cell cycle progression, the inhibition of angiogenesis(the formation of new blood vessels), the inhibition of metastasis (thespread of a tumour from its origin), the inhibition of cell migration(the spread of cancer cells to other parts of the body), the inhibitionof invasion (the spread of tumour cells into neighbouring normalstructures), or the promotion of cell apoptosis (programmed cell death).The compounds of the present invention may be used in the treatment ofthe cancers described herein, independent of the mechanisms discussedherein.

Treatment

The term “treatment,” as used herein in the context of treating adisorder, pertains generally to treatment of a human or an animal (e.g.,in veterinary applications), in which some desired therapeutic effect isachieved, for example, the inhibition of the progress of the disorder,and includes a reduction in the rate of progress, a halt in the rate ofprogress, alleviation of symptoms of the disorder, amelioration of thedisorder, and cure of the disorder. Treatment as a prophylactic measure(i.e., prophylaxis) is also included. For example, use with patients whohave not yet developed the disorder, but who are at risk of developingthe disorder, is encompassed by the term “treatment.”

For example, treatment includes the prophylaxis of cancer, reducing theincidence of cancer, alleviating the symptoms of cancer, etc.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of a compound, or a material, composition or dosage formcomprising a compound, which is effective for producing some desiredtherapeutic effect, commensurate with a reasonable benefit/risk ratio,when administered in accordance with a desired treatment regimen.

Combination Therapies

The term “treatment” includes combination treatments and therapies, inwhich two or more treatments or therapies are combined, for example,sequentially or simultaneously. For example, the compounds describedherein may also be used in combination therapies, e.g., in conjunctionwith other agents. Examples of treatments and therapies include, but arenot limited to, chemotherapy (the administration of active agents,including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs(e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery;radiation therapy; photodynamic therapy; gene therapy; and controlleddiets.

One aspect of the present invention pertains to a compound as describedherein, in combination with one or more (e.g., 1, 2, 3, 4, etc.)additional therapeutic agents, for example, agents or therapies thatregulate cell growth or survival or differentiation via a differentmechanism, thus treating several characteristic features of cancerdevelopment.

The particular combination would be at the discretion of the physicianwho would select dosages using his common general knowledge and dosingregimens known to a skilled practitioner.

The agents (i.e., the compound described herein, plus one or more otheragents) may be administered simultaneously or sequentially, and may beadministered in individually varying dose schedules and via differentroutes. For example, when administered sequentially, the agents can beadministered at closely spaced intervals (e.g., over a period of 5-10minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart,or even longer periods apart where required), the precise dosage regimenbeing commensurate with the properties of the therapeutic agent(s).

The agents (i.e., the compound described here, plus one or more otheragents) may be formulated together in a single dosage form, oralternatively, the individual agents may be formulated separately andpresented together in the form of a kit, optionally with instructionsfor their use.

Combination Therapies Employing DNA Damaging Agents

As discussed herein, in some embodiments, the PAPC compound is employedin combination with (e.g., in conjunction with) with one or more otheragents selected from: (a) a DNA topoisomerase I or II inhibitor; (b) aDNA damaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; (d) a microtubule targeted agent; and (e) ionising radiation.

When both a PAPC compound and one or more other agents are employed,they may be used (e.g., contacted, administered, etc.) in any order.Furthermore, they may be used (e.g., contacted, administered, etc.)together, as part of a single formulation, or separately, as separateformulations.

For example, in regard to methods of treatment employing both a PAPCcompound and one or more other agents, treatment with (e.g.,administration of) the PAPC compound may be prior to, concurrent with,or may follow, treatment with (e.g., administration of) the one or moreother agents, or a combination thereof.

In one embodiment, treatment with (e.g., administration of) a PAPCcompound is concurrent with, or follows, treatment with (e.g.,administration of) the one or more other agents.

In one embodiment, the one or more other agents is a DNA topoisomerase Ior II inhibitor; for example, Etoposide, Topotecan, Camptothecin,Irinotecan, SN-38, Doxorubicin, or Daunorubicin.

In one embodiment, the one or more other agents is a DNA damaging agent;for example, alkylating agents, platinating agents, or compounds thatgenerate free radicals; for example, Temozolomide, Cisplatin,Carboplatin, Oxaliplatin, Mitomycin C, Cyclophosphamide, BCNU, CCNU, orBleomycin.

In one embodiment, the one or more other agents is an antimetabolite orthymidylate synthase (TS) inhibitor; for example, 5-fluorouracil,hydroxyurea, Gemcitabine, Arabinosylcytosine, Fludarabine, Tomudex, orZD9331.

In one embodiment, the one or more other agents is a microtubuletargeted agent; for example, Paclitaxel, Docetaxel, Vincristine, orVinblastine.

In one embodiment, the one or more other agents is ionising radiation(e.g., as part of radiotherapy).

Other Uses

The PAPC compounds described herein may also be used as cell cultureadditives to inhibit CHK1 kinase function, e.g., to inhibit cellproliferation, etc.

The PAPC compounds described herein may also be used as part of an invitro assay, for example, in order to determine whether a candidate hostis likely to benefit from treatment with the compound in question.

The PAPC compounds described herein may also be used as a standard, forexample, in an assay, in order to identify other compounds, other CHK1kinase function inhibitors, other anti-proliferative agents, otheranti-cancer agents, etc.

Kits

One aspect of the invention pertains to a kit comprising (a) a PAPCcompound as described herein, or a composition comprising a PAPCcompound as described herein, e.g., preferably provided in a suitablecontainer and/or with suitable packaging; and (b) instructions for use,e.g., written instructions on how to administer the compound orcomposition.

In one embodiment, the kit further comprises one or more other agentsselected from: (a) a DNA topoisomerase I or II inhibitor; (b) a DNAdamaging agent; (c) an antimetabolite or thymidylate synthase (TS)inhibitor; and (d) a microtubule targeted agent.

The written instructions may also include a list of indications forwhich the active ingredient is a suitable treatment.

Routes of Administration

The PAPC compound or pharmaceutical composition comprising the PAPCcompound may be administered to a subject by any convenient route ofadministration, whether systemically/peripherally or topically (i.e., atthe site of desired action).

Routes of administration include, but are not limited to, oral (e.g., byingestion); buccal; sublingual; transdermal (including, e.g., by apatch, plaster, etc.); transmucosal (including, e.g., by a patch,plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., byeyedrops); pulmonary (e.g., by inhalation or insufflation therapy using,e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., bysuppository or enema); vaginal (e.g., by pessary); parenteral, forexample, by injection, including subcutaneous, intradermal,intramuscular, intravenous, intraarterial, intracardiac, intrathecal,intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal,intratracheal, subcuticular, intraarticular, subarachnoid, andintrasternal; by implant of a depot or reservoir, for example,subcutaneously or intramuscularly.

Preferably, the route of administration is oral, and the PAPC compoundor pharmaceutical composition comprising the PAPC compound isadministered to a subject orally.

The Subject/Patient

The subject/patient may be a chordate, a vertebrate, a mammal, aplacental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g.,a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), alagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig),ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., amonkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g.,gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus.

In one preferred embodiment, the subject/patient is a human.

Formulations

While it is possible for a PAPC compound to be administered alone, it ispreferable to present it as a pharmaceutical formulation (e.g.,composition, preparation, medicament) comprising at least one PAPCcompound, as described herein, together with one or more otherpharmaceutically acceptable ingredients well known to those skilled inthe art, including, but not limited to, pharmaceutically acceptablecarriers, diluents, excipients, adjuvants, fillers, buffers,preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,surfactants (e.g., wetting agents), masking agents, colouring agents,flavouring agents, and sweetening agents. The formulation may furthercomprise other active agents, for example, other therapeutic orprophylactic agents.

Thus, the present invention further provides pharmaceuticalcompositions, as defined above, and methods of making a pharmaceuticalcomposition comprising admixing at least one PAPC compound, as describedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts, for example, Remington's Pharmaceutical Sciences,18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbookof Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association thecompound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the compound with carriers (e.g.,liquid carriers, finely divided solid carrier, etc.), and then shapingthe product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations may suitably be in the form of liquids, solutions (e.g.,aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous),emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups,electuaries, mouthwashes, drops, tablets (including, e.g., coatedtablets), granules, powders, losenges, pastilles, capsules (including,e.g., hard and soft gelatin capsules), cachets, pills, ampoules,boluses, suppositories, pessaries, tinctures, gels, pastes, ointments,creams, lotions, oils, foams, sprays, mists, or aerosols.

Formulations may suitably be provided as a patch, adhesive plaster,bandage, dressing, or the like which is impregnated with one or morecompounds and optionally one or more other pharmaceutically acceptableingredients, including, for example, penetration, permeation, andabsorption enhancers. Formulations may also suitably be provided in theform of a depot or reservoir.

The compound may be dissolved in, suspended in, or admixed with one ormore other pharmaceutically acceptable ingredients. The compound may bepresented in a liposome or other microparticulate which is designed totarget the compound, for example, to blood components or one or moreorgans.

Formulations suitable for oral administration (e.g., by ingestion)include liquids, solutions (e.g., aqueous, non-aqueous), suspensions(e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water,water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders,capsules, cachets, pills, ampoules, boluses.

Formulations suitable for buccal administration include mouthwashes,losenges, pastilles, as well as patches, adhesive plasters, depots, andreservoirs. Losenges typically comprise the compound in a flavoredbasis, usually sucrose and acacia or tragacanth. Pastilles typicallycomprise the compound in an inert matrix, such as gelatin and glycerin,or sucrose and acacia. Mouthwashes typically comprise the compound in asuitable liquid carrier.

Formulations suitable for sublingual administration include tablets,losenges, pastilles, capsules, and pills.

Formulations suitable for oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),mouthwashes, losenges, pastilles, as well as patches, adhesive plasters,depots, and reservoirs.

Formulations suitable for non-oral transmucosal administration includeliquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g.,aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil),suppositories, pessaries, gels, pastes, ointments, creams, lotions,oils, as well as patches, adhesive plasters, depots, and reservoirs.

Formulations suitable for transdermal administration include gels,pastes, ointments, creams, lotions, and oils, as well as patches,adhesive plasters, bandages, dressings, depots, and reservoirs.

Tablets may be made by conventional means, e.g., compression ormoulding, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing in a suitable machine thecompound in a free-flowing form such as a powder or granules, optionallymixed with one or more binders (e.g., povidone, gelatin, acacia,sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers ordiluents (e.g., lactose, microcrystalline cellulose, calcium hydrogenphosphate); lubricants (e.g., magnesium stearate, talc, silica);disintegrants (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose); surface-active ordispersing or wetting agents (e.g., sodium lauryl sulfate);preservatives (e.g., methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,sorbic acid); flavours, flavour enhancing agents, and sweeteners.Moulded tablets may be made by moulding in a suitable machine a mixtureof the powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the compound therein using, forexample, hydroxypropylmethyl cellulose in varying proportions to providethe desired release profile. Tablets may optionally be provided with acoating, for example, to affect release, for example an enteric coating,to provide release in parts of the gut other than the stomach.

Ointments are typically prepared from the compound and a paraffinic or awater-miscible ointment base.

Creams are typically prepared from the compound and an oil-in-watercream base. If desired, the aqueous phase of the cream base may include,for example, at least about 30% w/w of a polyhydric alcohol, i.e., analcohol having two or more hydroxyl groups such as propylene glycol,butane-1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycoland mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the compoundthrough the skin or other affected areas. Examples of such dermalpenetration enhancers include dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the compound and an oily phase,which may optionally comprise merely an emulsifier (otherwise known asan emulgent), or it may comprise a mixture of at least one emulsifierwith a fat or an oil or with both a fat and an oil. Preferably, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabiliser. It is also preferred to include both an oiland a fat. Together, the emulsifier(s) with or without stabiliser(s)make up the so-called emulsifying wax, and the wax together with the oiland/or fat make up the so-called emulsifying ointment base which formsthe oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulfate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used.

Formulations suitable for intranasal administration, where the carrieris a liquid, include, for example, nasal spray, nasal drops, or byaerosol administration by nebuliser, include aqueous or oily solutionsof the compound.

Formulations suitable for intranasal administration, where the carrieris a solid, include, for example, those presented as a coarse powderhaving a particle size, for example, in the range of about 20 to about500 microns which is administered in the manner in which snuff is taken,i.e., by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose.

Formulations suitable for pulmonary administration (e.g., by inhalationor insufflation therapy) include those presented as an aerosol sprayfrom a pressurised pack, with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoromethane,dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.

Formulations suitable for ocular administration include eye dropswherein the compound is dissolved or suspended in a suitable carrier,especially an aqueous solvent for the compound.

Formulations suitable for rectal administration may be presented as asuppository with a suitable base comprising, for example, natural orhardened oils, waxes, fats, semi-liquid or liquid polyols, for example,cocoa butter or a salicylate; or as a solution or suspension fortreatment by enema.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the compound, such carriers as are known inthe art to be appropriate.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the compound isdissolved, suspended, or otherwise provided (e.g., in a liposome orother microparticulate). Such liquids may additionally contain otherpharmaceutically acceptable ingredients, such as anti-oxidants, buffers,preservatives, stabilisers, bacteriostats, suspending agents, thickeningagents, and solutes which render the formulation isotonic with the blood(or other relevant bodily fluid) of the intended recipient. Examples ofexcipients include, for example, water, alcohols, polyols, glycerol,vegetable oils, and the like. Examples of suitable isotonic carriers foruse in such formulations include Sodium Chloride Injection, Ringer'sSolution, or Lactated Ringer's Injection. Typically, the concentrationof the compound in the liquid is from about 1 ng/mL to about 10 μg/mL,for example from about 10 ng/mL to about 1 μg/mL. The formulations maybe presented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the PAPC compounds, and compositions comprising the PAPCcompounds, can vary from patient to patient. Determining the optimaldosage will generally involve the balancing of the level of therapeuticbenefit against any risk or deleterious side effects. The selecteddosage level will depend on a variety of factors including, but notlimited to, the activity of the particular PAPC compound, the route ofadministration, the time of administration, the rate of excretion of thePAPC compound, the duration of the treatment, other drugs, compounds,and/or materials used in combination, the severity of the disorder, andthe species, sex, age, weight, condition, general health, and priormedical history of the patient. The amount of PAPC compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the PAPC compound is in the range ofabout 10 μg to about 250 mg (more typically about 100 μg to about 25 mg)per kilogram body weight of the subject per day. Where the compound is asalt, an ester, an amide, a prodrug, or the like, the amountadministered is calculated on the basis of the parent compound and sothe actual weight to be used is increased proportionately.

EXAMPLES Chemical Synthesis

The following examples are provided solely to illustrate the presentinvention and are not intended to limit the scope of the invention, asdescribed herein.

General Synthetic Procedures

Reactions were carried out under N₂. Microwave reactions were carriedout using Biotage Initiator 60 or CEM microwave reactors. Flash silicachromatography was performed using Merck silica gel 60 (0.025-0.04 mm).Ion exchange chromatography was performed using Isolute Flash SCX-II(acidic) or Flash NH₂ (basic) resin cartridges. Gradient chromatographywas carried out on a Biotage SP1 automated flash chromatographypurification system. ¹H NMR spectra were recorded on a Bruker AMX500instrument at 500 MHz or a Bruker Avance instrument at 400 MHz usinginternal deuterium locks. Chemical shifts (δ) are reported relative toTMS (δ=0) and/or referenced to the solvent in which they were measured.Coupling constants (J) are reported in Hz. Combined HPLC-MS analyseswere recorded using either:

1. (LCT) a Waters Alliance 2795 separations module and Waters/MicromassLCT mass detector with electrospray ionization (+ve or −ve ion mode asindicated) with HPLC performed using Supelco DISCOVERY C18, 50 mm×4.6 mmor 30 mm×4.6 mm i.d. columns, or Agilent 6210 TOF HPLC-MS with aPhenomenex Gemini 3 μm C18 (3 cm×4.6 mm i.d.) column. Both were run at atemperature of 22° C. with gradient elution of 10-90% MeOH/0.1% aqueousformic acid at a flow rate of 1 mL/min and a run time of 3.5 or 4minutes as indicated. UV detection was at 254 nm and ionisation was bypositive or negative ion electrospray. Molecular weight scan range was50-1000 amu.2. (ZQ) a Micromass ZQ mass spectrometer/Waters Alliance 2795 HT HPLCwith a Phenomenex Gemini 5 μm, C18, 30 mm×4.6 mm i.d. column or WatersX-Bridge C18, 2.5 μm, 3.0×30 mm. Both were run at a temperature of 35°C. with a gradient elution of 5-95% (0.1% Ammonia in acetonitrile)/(0.1%Ammonia, 5% acetonitrile and 0.063% ammonium formate in water) at a flowrate of 2 mL/min and a run time of 4 or 6 minutes as indicated. UVdetection was at 220-400 nm using a Waters 996 photodiode array UVdetector and ionisation was by positive or negative ion electrospray.Molecular weight scan range was 80-1000 amu.

General Procedure i Coupling of 5-iodo-2-chloropyridine Intermediates to1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole

The appropriate 5-iodo-2-chloropyridine Intermediate I-8, or I-9 (1 eq)was dissolved in acetonitrile (7 mL solvent per 1 mmol of compound).1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1eq), tetrakis-(triphenylphosphine)palladium(0) (5 mol %) and sodiumcarbonate (1.5 eq) were added and the mixture was heated in a microwavereactor at 100° C. for 30 minutes (Intermediate I-8) or 15 minutes(Intermediate I-9). The reaction mixture was concentrated in vacuo ontosilica gel. Gradient chromatography (1-5% MeOH:CH₂Cl over 15 columnvolume and 5-10% over 8 column volume) gave the required 5-substitutedproduct.

General Procedure ii Coupling of 5-iodo-2-chloropyridine Intermediatesto ethynyltrimethylsilane or trimethyl((2-methylbut-3-yn-2-yl)oxy)silane

The appropriate 5-iodo-2-chloropyridine selected from Intermediates I-8,or I-9 (1 eq) 0.365 mmol) was dissolved in DMF (0.9 mL per 1 mmol ofcompound). Trimethyl(2-methylbut-3-yn-2-yloxy)silane orethynyltrimethylsilane (1.3 eq) was added as appropriate.Dichlorobis(triphenylphosphine)palladium(II) (6 mol %), copper(I) iodide(6 mol %) and triethylamine (18 eq) were added. The mixture was heatedin a microwave reactor at 120° C. for 10 minutes. The mixture was cooledand evaporated to dryness. Flash column chromatography on silica gel,eluting with ethyl acetate-hexane mixtures, gave the required5-substituted product.

General Procedure iii Coupling of 2-aminopyrazine Intermediates I-3, I-4or I-5 to 2-chloropyridine Intermediates

The appropriate 2-chloropyridine selected from Intermediates I-10, I-12,I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, or I-22 (1 eq) wasdissolved in toluene or dioxane (10 mL of solvent per 1 mmol ofcompound). The appropriate 2-aminopyrazine selected from IntermediatesI-3, I-4, or I-5 (1 eq) was added. Xantphos (20 mol %), cesium carbonate(2 eq) and tris(dibenzylideneacetone)-dipalladium(0) (10 mol %) wereadded. The mixture was heated in the microwave at 130° C. for 60minutes. The reaction mixture was diluted with MeOH (10 mL) and loadedonto a 2 g SCX-2 acidic ion exchange column. The column was flushed withMeOH (4×20 mL), followed by a solution of ammonia in MeOH (2 M; 4×20mL). The basic elutant was concentrated in vacuo onto silica gel.Gradient chromatography (1-10% MeOH: 1% NH₃ in CH₂Cl₂ over 15 columnvolume) gave the required coupled product. If further purification wasrequired to remove excess pyrazine starting material, then the materialwas subject to graduated preparative TLC (2-10% MeOH: 1% NH₃ in CH₂Cl₂).

General Procedure iv Coupling of 2-aminopyrazine Intermediates I-6 orI-7 to 2-chloropyridine Intermediates with N-Boc deprotection

The appropriate 2-chloropyridine selected from Intermediates I-12, I-15,I-16, or I-21 (1 eq) was dissolved in toluene or dioxane (10 mL ofsolvent per 1 mmol of compound). The appropriate 2-aminopyrazineselected from Intermediates I-6 or I-7 (1 eq) was added. Xantphos (20mol %), cesium carbonate (2 eq) andtris(dibenzylideneacetone)-dipalladium(0) (10 mol %) were added. Themixture was heated in the microwave at 130° C. for 60 minutes. Thereaction mixture was diluted with MeOH (10 mL) and loaded onto a 2 gSCX-2 acidic ion exchange column. The column was flushed with MeOH (4×20mL), followed by a solution of ammonia in MeOH (2 M; 4×20 mL). The basicelutant was concentrated in vacuo onto silica gel and subject togradient chromatography (1-10% MeOH: 1% NH₃ in CH₂Cl₂ over 15 columnvolume) to give the N-Boc protected coupled product. The N-Boc protectedcoupled product (1 eq) was dissolved in CH₂Cl₂ (140 mL of solvent per 1mmol of compound). Trifluoroacetic acid (470 eq) was added in oneportion and the solution was stirred at room temperature for 30 minutes.The solution was concentrated in vacuo onto silica gel. Gradientchromatography (5% MeOH:1% NH₃ in CH₂Cl over 5 column volume, then 5-20%over 15 column volume) gave the required N-deprotected product. Ifrequired, the product was further purified by graduated preparativethin-layer chromatography (2-10% MeOH: 1% NH₃ in CH₂Cl₂).

Intermediate I-1 (R)-1-(Dimethylamino)propan-2-ol

Dimethylamine 40% in water (11.39 mL, 90 mmol) was slowly added to(R)-propylene oxide (5.25 mL, 74.9 mmol) which had been cooled in an icebath. This solution was stirred at room temperature for 2 hours beforebeing extracted with CH₂Cl₂ (4×5 mL). The combined organic layers weredried over Na₂SO₄ and pure (R)-1-(dimethylamino)propan-2-ol (5.12 g,49.6 mmol, 40% yield) was isolated as a clear oil by distillation underreduced pressure (50 mbar).

¹H NMR (CDCl₃, 500 MHz) δ 3.82-3.76 (m, 1H), 3.40 (brs, 1H), 2.27 (s,6H), 2.25-2.21 (m, 1H), 2.16-2.12 (m, 1H), 1.12 (d, J=6.0, 3H).

Intermediate I-2 5-Amino-3-chloropyrazine-2-carbonitrile

2,6-Dichloropyrazine (2.89 g, 19.4 mmol) was stirred in aqueous NH₃(28%, 10 mL) and heated to 100° C. in a sealed tube for 18 hours. Thereaction mixture was cooled and the resultant precipitate was filtered.Trituration with water and then ether gave 6-chloropyrazin-2-amine as awhite solid (2.28 g, 17.6 mmol, 91% yield).

¹H NMR (d₆-DMSO, 400 MHz) δ 6.9 (brs, 2H), 7.70 (d, J=0.4, 1H), 7.80 (d,J=0.4, 1H); LC-MS (ZQ, 6 minutes) Rt=1.05 minutes; m/z (ESI+) 130 (M+H).

6-Chloropyrazin-2-amine (2.50 g, 19.3 mmol) was stirred in CH₂Cl₂ (60mL) at 0° C. N-Bromosuccinimide (2.92 g, 16.4 mmol) was added slowly andthe reaction mixture was stirred at 0° C. for 60 minutes. The reactionmixture was filtered through celite and concentrated to give a brownoil. Purification by flash chromatography, eluting with 0-25%EtOAc-hexanes, gave 5-bromo-6-chloropyrazin-2-amine as a yellow solid(1.69 g, 8.16 mmol, 42% yield).

¹H NMR (d₆-DMSO, 400 MHz) δ 7.1 (brs, 2H), 7.65 (s, 1H); LC-MS (ZQ, 4minutes) Rt=1.46 minutes; m/z (ESI−) 205 (M−H).

A mixture of 5-bromo-6-chloropyrazin-2-amine (1.00 g, 4.8 mmol), copper(I) iodide (914 mg, 4.8 mmol), 18-crown-6 (95 mg, 0.36 mmol) andtetrakis(triphenylphosphine)palladium (0) (83 mg, 0.072 mmol) wassuspended in dry DMF (20 mL) and a stream of nitrogen was passed throughfor 5 minutes. Potassium cyanide (312 mg, 4.8 mmol) was added and themixture was stirred at room temperature for 30 minutes, then refluxed at200° C. for 3 hours. The mixture was cooled, diluted with EtOAc andabsorbed onto silica gel (10 g). DMF was removed by evaporation. Theproduct was purified by flash chromatography, eluting with 1:1EtOAc-hexanes, to yield 5-amino-3-chloropyrazine-2-carbonitrile as ayellow solid (607 mg, 3.93 mmol, 82% yield).

¹H NMR (d₆ DMSO, 400 MHz) δ 7.87 (s, 1H), 8.1 (brs, 2H); LC-MS (ZQ, 4minutes) Rt=1.20 minutes; m/z (ESI−) 153 (M−H).

Intermediate I-3(R)-5-Amino-3-((1-(dimethylamino)propan-2-yl)oxy)pyrazine-2-carbonitrile

(R)-1-(Dimethylamino)propan-2-ol (0.667 g, 6.47 mmol) was added dropwiseto a suspension of NaH (60% in oil; 0.388 g, 9.71 mmol) in dioxane (16.2mL) and stirred for 30 minutes. 5-Amino-3-chloropyrazine-2-carbonitrile(Intermediate I-2) (1.00 g, 6.47 mmol) was added in one portion and themixture was heated at 90° C. for 14 hours.

After cooling, water (200 mL) was added and the solution was extractedwith Et₂O (4×100 mL), dried over MgSO₄, and the volatiles removed undervacuum. Gradient column chromatography eluting with MeOH: 1% NH₃ inCH₂Cl₂, gave(R)-5-amino-3-((1-(dimethylamino)propan-2-yl)oxy)pyrazine-2-carbonitrile(0.558 g, 2.52 mmol, 39% yield) as a yellow solid.

¹H NMR (500 MHz, CDCl₃) δ 7.54 (s, 1H), 5.42-5.35 (m, 1H), 5.31 (brs,2H), 2.76 (dd, J=7.5, 13.5, 1H), 2.52 (dd, J=4.0, 13.5, 1H), 2.37 (s,6H), 1.35 (d, J=6.5, 3H); LC-MS (LCT, 3.5 minutes) Rt=0.80 minutes; m/z(ESI) 222 (M+H).

Intermediate I-4(R)-5-Amino-3-((1-methylpyrrolidin-3-yl)oxy)pyrazine-2-carbonitrile

Prepared as described for Intermediate I-3 replacing(R)-1-(dimethylamino)propan-2-ol with (R)-1-methylpyrrolidin-3-ol.

¹H NMR (500 MHz, d₆-DMSO) δ 7.60 (brs, 2H), 7.51 (s, 1H), 5.36-5.28 (m,1H), 2.76 (dd, J=10.8, 6.1, 1H), 2.69 (ddd, J=8.2, 8.2, 5.3, 1H), 2.62(dd, J=10.8, 2.8, 1H), 2.37-2.21 (m, 2H), 2.26 (s, 3H), 1.85-1.77 (m,1H); LC-MS (LCT, 3.5 minutes) Rt=0.62 minutes; m/z (ESI) 220 (M+H).

Intermediate I-5(S)-5-Amino-3-((1-methylpyrrolidin-3-yl)oxy)pyrazine-2-carbonitrile

Prepared as described for Intermediate I-3 replacing(R)-1-(dimethylamino)propan-2-ol with (S)-1-methylpyrrolidin-3-ol.

¹H NMR (500 MHz, d₆-DMSO) δ 7.60 (brs, 2H), 7.51 (s, 1H), 5.36-5.28 (m,1H), 2.76 (dd, J=10.8, 6.1, 1H), 2.69 (ddd, J=8.2, 8.2, 5.3, 1H), 2.62(dd, J=10.8, 2.8, 1H), 2.37-2.21 (m, 2H), 2.26 (s, 3H), 1.85-1.77 (m,1H); LC-MS (LCT, 3.5 minutes) Rt=0.62 minutes; m/z (ESI) 220 (M+H).

Intermediate I-6 (R)-tert-Butyl3-((6-amino-3-cyanopyrazin-2-yl)oxy)pyrrolidine-1-carboxylate

Prepared as described for Intermediate I-3 replacing(R)-1-(dimethylamino)propan-2-ol with (R)-tert-butyl3-hydroxypyrrolidine-1-carboxylate. Isolated as a mixture of rotamers.¹H NMR (500 MHz, CDCl₃) δ7.61-7.58 (m, 1H), 5.49-5.44 (m, 1H), 5.35-5.30(m, 2H), 3.69-3.65 (m, 1H), 3.63-3.50 (m, 3H), 2.28-2.11 (m, 2H), 1.46(s, 9H); LC-MS (LCT, 3.5 minutes) Rt=2.67 minutes; m/z (ESI) 328 (M+Na).

Intermediate I-7 (S)-tert-Butyl3-((6-amino-3-cyanopyrazin-2-yl)oxy)pyrrolidine-1-carboxylate

Prepared as described for Intermediate I-3 replacing(R)-1-(dimethylamino)propan-2-ol with (S)-tert-butyl3-hydroxypyrrolidine-1-carboxylate.

Isolated as a mixture of rotamers. ¹H NMR (500 MHz, CDCl₃) δ7.61-7.58(m, 1H), 5.49-5.44 (m, 1H), 5.35-5.30 (m, 2H), 3.69-3.65 (m, 1H),3.63-3.50 (m, 3H), 2.28-2.11 (m, 2H), 1.46 (s, 9H); LC-MS (LCT, 3.5minutes) Rt=2.67 minutes; m/z (ESI) 328 (M+Na).

Intermediate I-8 2-Chloro-5-iodo-N-methylpyridin-4-amine

2-Chloro-5-iodopyridin-4-amine (2.0 g, 7.86 mmol) and paraformaldehyde(0.472 g, 15.7 mmol) were dissolved in AcOH (56.1 mL) and stirred for2.5 hours at 40° C. Sodium triacetoxyborohydride (3.66 g, 17.3 mmol) wasadded and the mixture was stirred at 40° C. for 1.5 hours. Furthersodium triacetoxyborohydride (3.66 g, 17.3 mmol) was added and themixture was stirred for a further 19 hours. The reaction mixture wasreduced in volume by half by evaporation in vacuo. Water was added tothe mixture, followed by basification with NaHCO₃. The mixture wasextracted with EtOAc (3×70 mL) and the combined organic layers weredried over MgSO₄. Silica was added and the solution was concentrated.Gradient chromatography, eluting with 5-10% EtOAc in c-Hex for 4 columnvolume and then 10% EtOAc in c-Hex for a further 11 column volume, gave2-chloro-5-iodo-N-methylpyridin-4-amine (1.65 g, 6.14 mmol, 78% yield)as a white crystalline powder.

¹H NMR (500 MHz, CDCl₃) δ 8.27 (s, 1H), 6.41 (s, 1H), 4.85 (brs, 1H),2.93 (d, J=5.0, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.90 minutes; m/z (ESI)268 (M+H).

Intermediate I-9 2-Chloro-5-iodo-4-methoxypyridine

To a solution of 2-chloro-4-methoxypyridine (0.5 g, 3.48 mmol) insulfuric acid (2.5 mL) was added N-iodosuccinimide (0.825 g, 3.48 mmol)portionwise at room temperature. The mixture was stirred at 55° C. for 2hours. The reaction mixture was poured into ice water (10 mL) and 8 MNaOH (20 mL) was added slowly, after which the dark brown solutionturned pale yellow. The aqueous layer was extracted with CH₂Cl₂ (2×20mL). The organic layers were washed with brine (10 mL) and concentratedin vacuo onto silica gel. Dry flash chromatography, eluting with 25%EtOAc:c-Hex, gave 2-chloro-5-iodo-4-methoxypyridine as a whitecrystalline solid (0.169 g, 0.760 mmol, 22% yield).

¹H NMR (500 MHz, d₆-DMSO) δ 8.52 (s, 1H), 7.19 (s, 1H), 3.96 (s, 3H);LC-MS (LCT, 4 minutes) Rt=2.56 minutes; m/z (ESI) 270 (M+H).

Intermediate I-10 Methyl 6-chloro-4-(methylamino)nicotinate

40% Methylamine in water (0.847 mL, 9.78 mmol) was added slowly over 5minutes at 0° C. to a solution of methyl 4,6-dichloronicotinate (0.40 g,1.94 mmol) in MeCN (6 mL). The solution was stirred at 0° C. for 30minutes then at room temperature for 2 hours. The reaction mixture wasconcentrated in vacuo onto silica gel. Gradient chromatography, elutingwith 5% EtOAc:c-Hex over 5 column volume and 5-50% over 15 columnvolume, gave methyl 6-chloro-4-(methylamino)nicotinate (278 mg, 1.386mmol, 71.4% yield) as a white solid.

¹H NMR (500 MHz, CDCl₃) δ 8.65 (s, 1H), 8.10 (brs, 1H), 6.54 (s, 1H),3.88 (s, 3H), 2.92 (d, J=5.1, 3H); LC-MS (LCT, 3.5 minutes) Rt=2.35minutes; m/z (ESI) 201 (M+H).

Intermediate I-11 Methyl 6-chloro-4-methoxynicotinate

Sodium methoxide powder (0.136 g, 2.52 mmol) was added slowly to astirred solution of methyl 4,6-dichloronicotinate (0.40 g, 1.94 mmol) inTHF (4 mL) at room temperature. The reaction mixture was stirred at roomtemperature for 18 hours, then concentrated in vacuo onto silica gel.Gradient chromatography, eluting with 5% EtOAc:c-Hex over 5 columnvolume and 5-50% over 15 column volume, gave methyl6-chloro-4-methoxynicotinate (218 mg, 1.08 mmol, 56% yield) as a whitecrystalline solid.

¹H NMR (500 MHz, CDCl₃) δ 8.71 (s, 1H), 6.92 (s, 1H), 3.97 (s, 3H), 3.90(s, 3H); LC-MS (LCT, 3.5 minutes) Rt=2.13 minutes; m/z (ESI) 202 (M+H).

Intermediate I-12 Methyl 6-chloro-4-(dimethylamino)nicotinate

Dimethylamine (1.23 mL, 9.71 mmol) was slowly added to a stirredsolution of methyl 4,6-dichloronicotinate (0.40 g, 1.94 mmol) in MeCN (6mL) at room temperature. The solution was stirred at room temperaturefor 18 hours then concentrated in vacuo onto silica gel. Gradientchromatography, eluting with 5% EtOAc:c-Hex over 5 column volume and5-50% over 15 column volume, gave methyl6-chloro-4-(dimethylamino)nicotinate (335 mg, 1.56 mmol, 80% yield) as awhite solid.

¹H NMR (500 MHz, d₆-DMSO) δ 8.21 (s, 1H), 6.85 (s, 1H), 3.83 (s, 3H),2.90 (s, 6H); LC-MS (LCT, 3.5 minutes) Rt=2.23 minutes; m/z (ESI) 215(M+H).

Intermediate I-13 2-Chloro-N-methyl-5-(trifluoromethyl)pyridin-4-amine

2 M Methylamine in MeOH (11.6 mL, 23.2 mmol) was added to2-chloro-4-iodo-5-(trifluoromethyl)pyridine (357 mg, 1.16 mmol) and themixture was heated in a microwave reactor at 130° C. for 1 hour. Themixture was concentrated in vacuo. Preparative thin-layerchromatography, eluting with 20% EtOAc:hexane, gave2-chloro-N-methyl-5-(trifluoromethyl)pyridin-4-amine (77 mg, 0.363 mmol,31% yield).

¹H NMR (500 MHz, d₆-DMSO) δ 8.17 (s, 1H), 6.90 (brs, 1H), 6.74 (s, 1H),2.81 (d, J=5, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.98 minutes; m/z (ESI)211 (M+H).

Intermediate I-14 2,5-Dichloro-N-methylpyridin-4-amine

N-Chlorosuccinimide (0.623 g, 4.67 mmol) was added to2-chloro-N-methyl-pyridin-4-amine (0.50 g, 3.89 mmol) and potassiumacetate (0.763 g, 7.78 mmol) in AcOH (25 mL) and the mixture was stirredat 80° C. for 1.25 hours. The mixture was cooled and concentrated invacuo. The concentrated mixture was diluted with water, neutralised withaqueous NaOH and extracted with EtOAc (x3). The organic extracts wereevaporated onto silica gel. Gradient chromatography, eluting with 10-50%EtOAc:c-Hex, gave 2,5-dichloro-N-methylpyridin-4-amine (0.114 g, 0.699mmol, 18% yield).

¹H NMR (500 MHz, CDCl₃) δ 8.02 (s, 1H), 6.52 (s, 1H), 4.95 (brs, 1H),2.96 (d, J=5, 3H); LC-MS (LCT, 4 minutes) Rt=2.17 minutes; m/z (ESI) 177(M+H).

Intermediate I-152-Chloro-N-methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine

Prepared from Intermediate I-8 and1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolefollowing General Procedure i.

¹H NMR (500 MHz, CDCl₃) δ 7.83 (s, 1H), 7.56 (s, 1H), 7.45 (s, 1H), 6.48(s, 1H), 4.68 (brs, 1H), 3.96 (s, 3H), 2.84 (d, J=5.1, 3H); LC-MS (LCT,3.5 minutes) Rt=1.09 minutes; m/z (ESI) 223 (M+H).

Intermediate I-162-Chloro-4-methoxy-5-(1-methyl-1H-pyrazol-4-yl)pyridine

Prepared from Intermediate I-9 and1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolefollowing General Procedure i.

¹H NMR (500 MHz, CDCl₃) δ 7.83 (s, 1H), 7.56 (s, 1H), 7.45 (s, 1H), 6.48(s, 1H), 4.68 (brs, 1H), 3.96 (s, 3H), 2.84 (d, J=5.1, 3H); LC-MS (LCT,3.5 minutes) Rt=1.09 minutes; m/z (ESI) 224 (M+H).

Intermediate I-172-Chloro-N,N-dimethyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine

DMF (2.99 mL) was slowly added to stirred sodium hydride (60% in oil; 51mg, 1.28 mmol) and2-chloro-N-methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine(Intermediate I-15) (104 mg, 0.467 mmol) at room temperature. Themixture was warmed to 80° C. for 10 minutes, followed by addition ofiodomethane (0.035 mL, 0.560 mmol). The mixture was stirred at 80° C.for 30 minutes, then cooled and diluted with saturated aqueous NaHCO₃(45 mL) and ethyl acetate (70 mL). After stirring for 10 minutes, theorganic layer was separated and the aqueous layer was extracted withethyl acetate (2×70 mL). The combined organic layers were evaporatedonto silica gel. Gradient chromatography, eluting with 1-10% MeOH: 1%NH₃ in CH₂Cl₂ over 10 column volume, gave2-chloro-N,N-dimethyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine (102mg, 0.431 mmol, 92% yield).

¹H NMR (500 MHz, CDCl₃) δ 8.03 (s, 1H), 7.67 (s, 1H), 7.57 (s, 1H), 6.77(s, 1H), 3.96 (s, 3H), 2.72 (s, 6H).

Intermediate I-182-Chloro-N-ethyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine

A mixture of 2-chloro-5-iodopyridin-4-amine (262 mg, 1.03 mmol), excessparaformaldehyde (618 mg, 21 mmol) and AcOH (10.3 mL) was stirred at 40°C. for 15 minutes, followed by addition of excess sodiumtriacetoxyborohydride (4.8 g, 23 mmol). After stirring for 2.5 hours,further paraformaldehyde (236 mg, 7.86 mmol) and sodiumtriacetoxyborohydride (1.83 g, 8.63 mmol) were added. After 18 hours,the mixture was diluted with water and basified with NaHCO₃. The mixturewas extracted with EtOAc (3×30 mL). The combined organic extracts weredried and evaporated onto silica. Gradient chromatography, eluting with5-10% EtOH: CH₂Cl₂ over 17 column volume, gave2-chloro-N-ethyl-5-iodopyridin-4-amine (291 mg, 1.03 mmol, 100% yield).LC-MS (LCT, 3.5 minutes) Rt=2.56 minutes; m/z (ESI) 282 (M+H). Thematerial was reacted with1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazolefollowing General Procedure i to give2-chloro-N-ethyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-4-amine.

¹H NMR (500 MHz, CDCl₃) δ 7.84 (s, 1H), 7.59 (s, 1H), 7.47 (s, 1H), 6.50(s, 1H), 4.60 (brs, 1H), 3.99 (s, 3H), 3.19 (2H, q, J=7.2), 1.25 (t,J=7.2, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.35 minutes; m/z (ESI) 237(M+H).

Intermediate I-192-Chloro-N-methyl-5-((trimethylsilyl)ethynyl)pyridin-4-amine

Prepared from Intermediate I-8 and ethynyltrimethylsilane followingGeneral Procedure ii.

¹H NMR (500 MHz, CDCl₃) δ 8.11 (s, 1H), 6.47 (s, 1H), 5.15 (brs, 1H),2.97 (d, J=5, 3H), 0.3 (9H, s); LC-MS (LCT, 4 minutes) Rt=3.13 minutes;m/z (ESI) 239 (M+H).

Intermediate I-202-Chloro-N-methyl-5-(3-methyl-3-((trimethylsilyl)oxy)but-1-yn-1-yl)pyridin-4-amine

Prepared from Intermediate I-8 andtrimethyl(2-methylbut-3-yn-2-yloxy)silane following General Procedureii.

¹H NMR (500 MHz, CDCl₃) δ 8.04 (s, 1H), 6.46 (s, 1H), 5.00 (brs, 1H),2.94 (d, J=5, 3H), 1.62 (6H, s), 0.22 (s, 9H); LC-MS (LCT, 3.5 minutes)Rt=2.81 minutes; m/z (ESI) 297 (M+H).

Intermediate I-212-Chloro-4-methoxy-5-(3-methyl-3-((trimethylsilyl)oxy)but-1-yn-1-yl)pyridine

Prepared from Intermediate I-9 andtrimethyl(2-methylbut-3-yn-2-yloxy)silane following General Procedureii.

¹H NMR (500 MHz, CDCl₃) δ 8.25 (s, 1H), 6.82 (s, 1H), 3.92 (s, 3H), 1.60(6H, s), 0.23 (s, 9H); LC-MS (LCT, 4 minutes) Rt=3.23 minutes; m/z (ESI)298 (M+H).

Intermediate I-22 2-Chloro-5-cyclopropyl-N-methylpyridin-4-amine

2-Chloro-5-iodo-N-methylpyridin-4-amine (Intermediate I-8) (30 mg, 0.112mmol), tetrakis(triphenylphosphine) palladium (0) (6.5 mg, 5.59 μmol)and 0.5 M aqueous sodium carbonate solution (290 μL, 0.145 mmol) wereadded to 2-cyclopropyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (61 μL,0.335 mmol) in MeCN. The mixture was heated at 130° C. in a microwavereactor for 1 hour. The mixture was concentrated in vacuo. Preparativethin layer chromatography, eluting with 1% NH₃, 6% MeOH in CH₂Cl₂, gave2-chloro-5-cyclopropyl-N-methylpyridin-4-amine (10 mg, 0.055 mmol, 49%yield) as a white powder.

¹H NMR (500 MHz, CDCl₃) δ 7.72 (s, 1H), 6.34 (s, 1H), 4.82 (brs, 1H),2.85 (s, 3H), 1.37-1.34 (m, 1H), 0.85-0.82 (m, 2H), 0.49-0.46 (m, 2H);LC-MS (LCT, 4 minutes) Rt=1.23 minutes; m/z (ESI) 183 (M+H).

Compound PAPC-A-01(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((4-methoxy-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-16 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃/CD₃OD) δ 9.14 (s, 1H), 8.35 (s, 1H), 7.83 (s,1H), 7.76 (s, 1H), 7.06 (s, 1H), 5.87-5.75 (m, 1H), 4.00 (s, 3H), 3.94(s, 3H), 3.35 (dd, J=13.7, 6.7, 1H), 3.21 (d, J=13.7, 1H), 2.87 (s, 6H),1.53 (d, J=6.4, 3H); LC-MS (LCT, 3.5 minutes) Rt=2.11 minutes; m/z (ESI)409 (M+H).

Compound PAPC-A-02(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((5-(1-methyl-1H-pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-15 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.23 (brs, 1H), 8.23 (s, 1H), 7.85 (s, 1H),7.58 (s, 1H), 7.47 (s, 1H), 7.03 (s, 1H), 5.48-5.37 (m, 1H), 4.72 (q,J=5, 1H), 3.98 (s, 3H), 2.90 (d, J=5, 3H), 2.74 (dd, J=13.4, 7.2, 1H),2.51 (dd, J=13.4, 4.4, 1H), 2.31 (s, 6H), 1.41 (d, J=6.3, 3H); LC-MS(LCT, 3.5 minutes) Rt=1.09 minutes; m/z (ESI) 408 (M+H).

Compound PAPC-A-03(R)-5-((4-(Dimethylamino)-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)-3-((1-(dimethylamino)propan-2-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-17 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.61 (brs, 1H), 8.23 (s, 1H), 8.04 (s, 1H),7.69 (s, 1H), 7.57 (s, 1H), 7.29 (s, 1H), 5.50-5.39 (m, 1H), 3.97 (s,3H), 2.76 (s, 6H), 2.73 (dd, J=13.4, 7.3, 1H), 2.50 (dd, J=13.4, 4.4,1H), 2.30 (s, 6H), 1.40 (d, J=6.3, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.59minutes; m/z (ESI) 418 (M+H).

Compound PAPC-A-04(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((4-(ethylamino)-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-18 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.29 (s, 1H), 7.86 (s, 1H), 7.61 (s, 1H), 7.50(s, 1H), 7.07 (s, 1H), 5.50-5.47 (m, 1H), 4.63-4.61 (m, 1H), 4.02 (s,3H), 3.27-3.23 (m, 2H), 2.84-2.80 (m, 1H), 2.62-2.59 (m, 1H), 2.39 (s,6H), 1.43 (d, J=6.3, 3H), 1.30 (t, J=7.3, 3H); LC-MS (LCT, 3.5 minutes)Rt=1.22 minutes; m/z (ESI) 422 (M+H).

Compound PAPC-A-05(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((4-(methylamino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-13 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CD₃OD) δ 8.60 (s, 1H), 8.16 (s, 1H), 6.98 (s, 1H),5.59-5.53 (m, 1H), 2.94 (s, 3H), 2.82 (dd, J=13.7, 8, 1H), 2.51 (dd,J=13.7, 5, 1H), 2.33 (s, 6H), 1.42 (d, J=6, 3H); LC-MS (LCT, 3.5minutes) Rt=1.75 minutes; m/z (ESI) 396 (M+H).

Compound PAPC-A-06(R)-5-((5-Chloro-4-(methylamino)pyridin-2-yl)amino)-3-((1-(dimethylamino)propan-2-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-14 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.22 (s, 1H), 8.02 (brs, 1H), 7.98 (s, 1H),7.01 (s, 1H), 5.48-5.36 (m, 1H), 4.97 (brq, J=4.2, 1H), 2.98 (d, J=5.1,3H), 2.74 (dd, J=13.3, 7.3, 1H), 2.51 (dd, J=13.3, 4.3, 1H), 2.31 (s,6H), 1.41 (d, J=6.3, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.77 minutes; m/z(ESI) 362 (M+H).

Compound PAPC-A-07(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((5-ethynyl-4-(methylamino)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-19 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.28 (s, 1H), 8.13 (s, 1H), 7.80 (brs, 1H),6.94 (s, 1H), 5.45-5.40 (m, 1H), 5.21-5.19 (m, 1H), 3.49 (s, 1H), 3.00(s, 3H), 2.76-2.73 (m, 1H), 2.54-2.52 (m, 1H), 2.34 (s, 6H), 1.43 (d,J=6, 3H); LC-MS (LCT, 4 minutes) Rt=1.53 minutes; m/z (ESI) 352 (M+H).

Compound PAPC-A-08(R)-3-((1-(Dimethylamino)propan-2-yl)oxy)-5-((5-(3-hydroxy-3-methylbut-1-yn-1-yl)-4-(methylamino)pyridin-2-yl)amino)pyrazine-2-carbonitrile

Prepared from Intermediate I-20 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CD₃OD) δ 8.46 (s, 1H), 7.95 (s, 1H), 6.92 (s, 1H),5.59-5.55 (m, 1H), 2.97 (s, 3H), 2.85 (dd, J=13, 10, 1H), 2.60 (dd,J=13, 5, 1H), 2.35 (s, 6H), 1.61 (s, 6H), 1.43 (d, J=6, 3H); LC-MS (LCT,3.5 minutes) Rt=1.43 minutes; m/z (ESI) 410 (M+H).

Compound PAPC-A-09(R)-5-((5-Cyclopropyl-4-(methylamino)pyridin-2-yl)amino)-3-((1-(dimethylamino)propan-2-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-22 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.11 (s, 1H), 7.71 (s, 1H), 6.91 (s, 1H),5.39-5.37 (m, 1H), 4.92 (brs, 1H), 2.93-2.29 (m, 3H), 2.73-2.68 (m, 1H),2.49 (dd, J=13.3, 3.9, 1H), 2.27 (s, 6H), 1.40-1.38 (m, 1H), 1.35 (d,J=6, 3H), 0.86-0.84 (m, 2H), 0.50-0.48 (m, 2H); LC-MS (LCT, 4 minutes)Rt=1.40 minutes; m/z (ESI) 368 (M+H).

Compound PAPC-A-10 (R)-Methyl6-((5-cyano-6-((1-(dimethylamino)propan-2-yl)oxy)pyrazin-2-yl)amino)-4-(methylamino)nicotinate

Prepared from Intermediate I-10 and Intermediate I-3 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.68 (s, 1H), 8.26 (s, 1H), 8.15 (d, J=5, 1H),7.03 (s, 1H), 5.50-5.39 (m, 1H), 3.88 (s, 3H), 2.97 (d, J=5, 3H), 2.78(dd, J=13.4, 7.3, 1H), 2.55 (dd, J=13.4, 4.2, 1 H), 2.34 (s, 6H), 1.42(d, J=6.3, 3H); LC-MS (LCT, 3.5 minutes) Rt=1.84 minutes; m/z (ESI) 386(M+H).

Compound PAPC-B-01(R)-5-((4-Methoxy-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)-3-(pyrrolidin-3-yloxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-16 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.51 (s, 1H), 8.45 (s, 1H), 8.13 (s, 1H),7.93 (d, J=0.6, 1H), 7.60 (s, 1H), 5.61-5.56 (m, 1H), 3.98 (s, 3H), 3.88(s, 3H), 3.23 (dd, J=12.7, 5.5, 1H), 3.06-2.97 (m, 2H), 2.94-2.87 (m,1H), 2.16-2.09 (m, 1H), 1.99-1.92 (m, 1H); LC-MS (LCT, 3.5 minutes)Rt=2.02 minutes; m/z (ESI) 393 (M+H).

Compound PAPC-B-02(R)-5-((4-Methoxy-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2-yl)amino)-3-((1-methylpyrrolidin-3-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-16 and Intermediate I-4 following GeneralProcedure iii.

¹H-NMR (500 MHz, CDCl₃) δ 8.39 (brs, 1H), 8.37 (s, 1H), 8.32 (s, 1H),7.85 (s, 1H), 7.78 (s, 1H), 7.36 (s, 1H), 5.51-5.47 (m, 1H), 4.01 (s,3H), 3.96 (s, 3H), 3.15 (dd, J=10.7, 6.3, 1H), 2.73-2.62 (m, 3H), 2.39(s, 3H), 2.38-2.30 (m, 1H), 2.12-2.05 (m, 1H); LC-MS (LCT, 3.5 minutes)Rt=2.01 minutes; m/z (ESI) 407 (M+H).

Compound PAPC-B-03(R)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)-3-(pyrrolidin-3-yloxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-15 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 10.46 (brs, 1H), 8.50 (s, 1H), 7.89 (s, 1H),7.84 (s, 1H), 7.60 (s, 1H), 7.04 (s, 1H), 5.84 (q, J=4.7, 1H), 5.61-5.55(m, 1H), 3.88 (s, 3H), 3.25 (dd, J=12.7, 5.5, 1H), 3.07-3.00 (m, 2H),2.93 (ddd, J=10.9, 8.1, 4.7, 1H), 2.78 (d, J=4.8, 3H), 2.17-2.10 (m,1H), 2.01-1.93 (m, 1H); LC-MS (LCT, 3.5 minutes) Rt=1.24 minutes; m/z(ESI) 392 (M+H).

Compound PAPC-B-04(S)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)-3-(pyrrolidin-3-yloxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-15 and Intermediate I-7 following GeneralProcedure iv.

H NMR (500 MHz, d₆-DMSO) δ 10.46 (brs, 1H), 8.50 (s, 1H), 7.89 (s, 1H),7.84 (s, 1H), 7.60 (s, 1H), 7.04 (s, 1H), 5.84 (q, J=4.7, 1H), 5.61-5.55(m, 1H), 3.88 (s, 3H), 3.25 (dd, J=12.7, 5.5, 1H), 3.07-3.00 (m, 2H),2.93 (ddd, J=10.9, 8.1, 4.7, 1H), 2.78 (d, J=4.8, 3H), 2.17-2.10 (m,1H), 2.01-1.93 (m, 1H), LC-MS (LCT, 3.5 minutes) Rt=1.24 minutes; m/z(ESI) 392 (M+H).

Compound PAPC-B-05(R)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)-3-((1-methylpyrrolidin-3-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-15 and Intermediate I-4 following GeneralProcedure iii.

¹H NMR (500 MHz, CD₃OD) δ 8.49 (s, 1H), 7.82 (s, 1H), 7.78 (s, 1H), 7.61(s, 1H), 6.85 (s, 1H), 5.62-5.60 (m, 1H), 3.97 (s, 3H), 3.05-3.02 (m,1H), 2.88 (3H, s), 2.87-2.85 (m, 2H), 2.59-2.55 (m, 1H), 2.48-2.43 (m,1H), 2.42 (s, 3H), 2.10-2.08 (m, 1H); LC-MS (LCT, 4 minutes) Rt=1.20minutes; m/z (ESI) 406 (M+H).

Compound PAPC-B-06(S)-5-((5-(1-Methyl-1H-pyrazol-4-yl)-4-(methylamino)pyridin-2-yl)amino)-3-((1-methylpyrrolidin-3-yl)oxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-15 and Intermediate I-5 following GeneralProcedure iii.

¹H NMR (500 MHz, CDCl₃) δ 8.33 (s, 1H), 7.86 (s, 1H), 7.59 (s, 1H), 7.50(s, 1H), 6.98 (s, 1H), 5.56-5.54 (m, 1H), 4.78-4.77 (m, 1H), 4.00 (s,3H), 3.29 (dd, J=11, 6.2, 1H), 2.92 (s, 3H), 2.80-2.78 (m, 1H), 2.74(dd, J=11, 3.5, 1H), 2.47 (s, 3H), 2.41-2.37 (m, 1H), 2.16-2.14 (m, 1H);LC-MS (LCT, 4 minutes) Rt=1.20 minutes; m/z (ESI) 406 (M+H).

Compound PAPC-B-07(R)-5-((5-(3-Hydroxy-3-methylbut-1-yn-1-yl)-4-methoxypyridin-2-yl)amino)-3-(pyrrolidin-3-yloxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-21 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.51 (s, 1H), 8.21 (s, 1H), 7.47 (s, 1H),5.62 (ddd, J=5.5, 5.5, 2.8, 1H), 5.41 (brs, 1H), 3.92 (s, 3H), 3.38 (dd,J=13, 5.3, 1H), 3.25 (d, J=13, 1H), 3.18-3.09 (m, 2H), 2.25-2.16 (m,1H), 2.14-2.07 (m, 1H), 1.47 (s, 6H); LC-MS (LCT, 3.5 minutes) Rt=1.84minutes; m/z (ESI) 395 (M+H).

Compound PAPC-B-08(R)-5-((5-(3-Hydroxy-3-methylbut-1-yn-1-yl)-4-(methylamino)pyridin-2-yl)amino)-3-(pyrrolidin-3-yloxy)pyrazine-2-carbonitrile

Prepared from Intermediate I-20 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.62 (s, 1H), 7.96 (s, 1H), 6.71 (s, 1H),5.76 (brs, 1H), 3.49-3.48 (m, 2H), 3.45-3.35 (m, 1H), 2.95 (s, 3H),2.35-2.32 (m, 2H), 1.60 (s, 6H) (2H obscured by water); LC-MS (LCT, 4minutes) Rt=1.59 minutes; m/z (ESI) 394 (M+H).

Compound PAPC-B-09 (R)-Methyl6-((5-cyano-6-(pyrrolidin-3-yloxy)pyrazin-2-yl)amino)-4-(methoxy)nicotinate

Prepared from Intermediate I-11 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.63 (s, 1H), 8.54 (s, 1H), 7.56 (s, 1H),5.62-5.60 (m, 1H), 3.94 (s, 3H), 3.79 (s, 3H), 3.18-2.98 (m, 4H),2.21-2.12 (m, 1H), 2.07-1.99 (m, 1H); LC-MS (LCT, 3.5 minutes) Rt=2.04minutes; m/z (ESI) 371 (M+H).

Compound PAPC-B-10 (R)-Methyl6-((5-cyano-6-(pyrrolidin-3-yloxy)pyrazin-2-yl)amino)-4-(methylamino)nicotinate

Prepared from Intermediate I-10 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.58 (s, 1H), 8.38 (s, 1H), 8.01 (q, J=4.7,1H), 7.22 (s, 1H), 5.62-5.51 (m, 1H), 3.81 (s, 3H), 3.18 (dd, J=12.6,5.5, 1H), 3.01-2.92 (m, 2H), 2.90 (d, J=4.9, 3H), 2.85 (ddd, J=10.8,8.0, 4.8, 1H), 2.14-2.03 (m, 1H), 1.95-1.87 (m, 1H); LC-MS (LCT, 3.5minutes) Rt=1.64 minutes; m/z (ESI) 370 (M+H).

Compound PAPC-B-11 (R)-Methyl6-((5-cyano-6-(pyrrolidin-3-yloxy)pyrazin-2-yl)amino)-4-(dimethylamino)nicotinate

Prepared from Intermediate I-12 and Intermediate I-6 following GeneralProcedure iv.

¹H NMR (500 MHz, d₆-DMSO) δ 8.56 (s, 1H), 8.35 (s, 1H), 7.20 (s, 1H),5.68-5.59 (m, 1H), 3.81 (s, 3H), 3.39 (dd, J=13.1, 5.2, 1H), 3.31 (bs,1H), 3.27 (d, J=13.1, 1H), 3.19-3.12 (m, 2H), 2.91 (s, 6H), 2.23-2.18(m, 1H), 2.15-2.08 (m, 1H); LC-MS (LCT, 3.5 minutes) Rt=1.70 minutes;m/z (ESI) 384 (M+H).

Biological Methods

Assay 1a: Determination of Inhibitor Potency Vs. CHK1 in DELFIA AssayFormat

CHK1 kinase function was measured in a DELFIA® assay in order to monitorphosphorylation of a CDC25C peptide using a specific phospho antibody.The enzyme reaction was carried out in polypropylene plates (Greiner)using a reaction mix (25 μL) containing enzyme and peptide mix (CHK1, 1nM; Biotin-KKKVSRSGLYRSPSMPENLNRPR, 1 μM or 15 μL), ATP (30 μM or 5 μL)and either DMSO (2.5%) or test compound (5 μL) diluted to a give a rangeof concentrations (from 0 to 100 μM in 2.5% DMSO, final concentrations)in assay buffer (40 mM Tris, 40 mM NaCl, 2 mM MgCl₂, 1 mM DTT and 0.1%Tween 20). The reaction mixture was incubated for 30 minutes at roomtemperature and then stopped by the addition of buffer (125 μL)containing 40 mM EDTA, 0.05% Tween 20, 0.1% BSA in TBS (10× concentrate,Sigma). An aliquot (100 μL) of the stopped reaction mixture wastransferred to a black neutravidin-coated plate (Perbio) and incubatedfor 1 hour on a shaker (Titertek, Flow Laboratories) at roomtemperature. The plates were washed four times with wash buffer (25 mMTris (pH 8), 150 mM NaCl, and 0.1% Tween 20) (WellWash4, Thermo LifeSciences) and incubated for 1 hour as before with an antibody mixture(100 μL) consisting of anti-phospho CDC25C (1.25 nM, #9528, CellSignalling Technology) and europium-labelled anti-rabbit IgG (0.3 μg/mL,AD0105, PerkinElmer Life Sciences) diluted in DELFIA assay buffer(PerkinElmer Life Sciences). The plates were washed a further four timeswith wash buffer before the addition of enhancement solution (100μL/well, PerkinElmer Life Sciences). The plate was read on a Victor21420 multilabel counter (Perkin Elmer Life Sciences) using atime-resolved measurement mode reading fluorescence at 615 nm. Theconcentration of test compound required to inhibit enzyme activity by50% was calculated (IC₅₀).

Assay 1B: Determination of Inhibitor Potency Vs. CHK1 in Caliper AssayFormat

CHK1 kinase activity was measured in a microfluidic assay that monitorsthe separation of a phosphorylated product from its substrate. The assaywas run on an EZ Reader II (Caliper Life Sciences Ltd, Runcorn, UK)using separation buffer (#760367 Caliper LS) containing CR-8 (500 nM,#760278, Caliper LS). An ECHO® 550 (Labcyte Inc™) acoustic dispenser wasused to generate duplicate 8 pt dilution curves directly into 384polypropylene assay plates (Greiner Bio-One, Gloucestershire, UK). Foreach test compound a 50 μM stock concentration in 100% DMSO was used.The total amount of DMSO dispensed per well was 250 nL to give a finalassay concentration of 2.5% DMSO and test compound concentrations in therange 0.5-1000 nM. To this assay plate, 6 μL CHK1 (2 nM finalconcentration, in-house protein preparation), 2 μL peptide 10(5-FAM-KKKVSRSGLYRSPSMPENLNRPR-COOH, 1.5 μM final concentration, #760354Caliper LS) and 2 μL ATP (90 μM final concentration) all diluted inkinase buffer (HEPES 50 mM, NaN₃ 0.02%, BSA 0.01%, sodium orthovanadate0.1 mM, DTT 1 mM, MgCl₂ 2 mM, Tween 20 0.1%) were added. The plate wassealed and centrifuged (1 minutes, 1000 rpm) before incubation for onehour at room temperature. The reaction was stopped by the addition ofseparation buffer (90 μL). The plate was read on an EZ Reader II, usinga 12-sipper chip (760137-0372R, Caliper LS) with instrument settings of−1.5 psi and 1750 ΔV. The percentage conversion of product fromsubstrate was generated automatically and the percentage inhibition wascalculated relative to blank wells (containing no enzyme and 2.5% DMSO)and total wells (containing all reagents and 2.5% DMSO). IC₅₀ valueswere calculated in GraphPad Prism5 using a non linear regression fit ofthe log(inhibitor) vs. response with variable slope equation.

Assay 2: Determination of Inhibitor Selectivity for Inhibition of CHK1Vs. CHK2

In vitro CHK2 kinase activity was measured in a DELFIA® assay thatmonitors phosphorylation of a CDC25C peptide using a specific phosphoantibody. The enzyme reaction was carried out in 96-well polypropyleneplates (Greiner). The reaction mix (total volume 25 μL) contained enzymeand peptide mix (15 μL) (containing CHK2, prepared in-house, 1 nM;Biotin-KKKVSRSGLYRSPSMPENLNRPR, 1 μM), ATP (30 μM, 5 μL) and either DMSO(2.5%) or test compound (5 μL) diluted to a give a range ofconcentrations (0-100 μM in 2.5% DMSO, final concentrations) in assaybuffer (40 mM HEPES (pH 7.4), 40 mM KCl, 2 mM MgCl₂, 10 mM DTT and 0.02%Tween 20). The reaction mixture was incubated for 30 minutes at roomtemperature and stopped by the addition of buffer (125 μL) containing 40mM EDTA, 0.05% Tween 20, 0.1% BSA in TBS (10× concentrate, Sigma). Analiquot (100 μL) of the reaction mix was transferred to a blackneutravidin-coated 96-well plate (Perbio) and incubated for 1 hour on ashaker (Titertek, Flow Laboratories) at room temperature. The plateswere washed four times with wash buffer (25 mM Tris (pH 8), 150 mM NaCland 0.1% Tween 20) (WellWash4, Thermo Life Sciences) and incubated for 1hour as before with antibody mix (100 μL) consisting of anti-phosphoCDC25C (diluted 1/4000 equivalent to 0.35 nM-1.25 nM, #9528, CellSignalling Technology) and europium-labelled anti rabbit IgG, (0.3μg/mL, AD0105, PerkinElmer Life Sciences) diluted in DELFIA® assaybuffer (PerkinElmer Life Sciences). The plates were washed a furtherfour times with wash buffer before the addition of enhancement solution(100 μL/well, PerkinElmer Life Sciences). The plate was read on aVictor2 1420 multi label counter (PerkinElmer Life Sciences) using atime-resolved measurement mode reading fluorescence at 615 nM. Theconcentration of test compound required to inhibit enzyme activity by50% was calculated (IC₅₀).

For each test compound, the ratio of the IC₅₀ from the CHK2 kinaseactivity assay to the IC₅₀ from the CHK1 kinase activity assay (i.e.,CHK2 IC₅₀/CHK1 IC₅₀) was used to define the selectivity for inhibitionof CHK1 vs. CHK2.

Assay 3: Mitosis Inhibition Assay (MIA)

Checkpoint abrogation by CHK1 kinase function inhibitors in combinationwith genotoxic agents was assessed using a europium based ELISA assaydesigned to quantify the number of cells trapped in mitosis aftertreatment with a genotoxic agent (to induce G2 arrest) followed by atest compound in combination with nocodazole to abrogate this arrest.HT29 cells were seeded at 104 cells per well into 96-well plates in avolume of 160 μL and left to attach for 36 hours. Etoposide (10 mM stockin DMSO) was diluted in medium to 250 μM and then 40 μL was added toappropriate wells to give a final concentration of 50 μM and incubatedfor 1 hour. This treatment had previously been optimised to induce a G2arrest in 80% of cells 16 hours following treatment. After genotoxicdrug exposure, the medium was removed and replaced with fresh medium(160 μL). Cells were either untreated (untreated control or etoposidepre-treatment alone), exposed to nocodazole following etoposide pretreatment or nocodazole alone (100 ng/mL final concentration), orexposed to increasing concentrations of test compound (from 200 μM to0.01 nM final concentration) in combination with nocodazole (100 ng/mLfinal concentration). Test compounds were added in 40 μL aliquots usingquadruplicate wells for each dose. After 21 hours exposure, the mediumwas removed and cells were fixed in 4% formaldehyde in phosphatebuffered saline (PBS, pH 7.4, pre-cooled to 4° C.) for 30 minutes at 4°C., followed by 100% methanol (pre-cooled to −20° C.) for 10 minutes atambient temperature. Wells were washed with PBS and blocked with 5%dried milk (Marvel) in Tris-buffered saline (TBS, pH 7.4) at 37° C. for30 minutes. Each well was washed three times with water containing 0.1%Tween 20. Primary antibody (MPM-2, Upstate cat#05-368, 1 μg/mL in 5%milk in TBS) was added to each well and incubated overnight with shakingat 4° C. Primary antibody was removed and wells were washed with watercontaining 0.1% Tween 20. The secondary antibody (europium labelledanti-mouse, Perkin-Elmer cat# AD0124, 333 ng/mL in assay bufferPerkin-Elmer cat#1244-111) was added to each well and incubated at 37°C. for 1 hour. Each well was washed with water 0.1% containing Tween 20and treated with enhancement solution (Perkin-Elmer cat#1244-105).Europium emissions were counted on a Wallac, Victor2 counter(Perkin-Elmer, Bucks UK). Appropriate controls were included and resultswere expressed as the concentration of test compound required to allow50% of cells to enter mitosis (MIA IC₅₀).

Estimation of Comparative Oral Bioavailability by Limited Sampling InVivo Pharmacokinetics

Female BALB/c mice (6 weeks old) (Charles River UK Ltd, Margate, UK)were kept in a controlled environment with food and sterilized wateravailable ad libitum. Animals weighed 20±2 g at the time of experiment.All procedures were conducted in accordance with the local and nationalguidelines for animal experimentation. Dosing solutions were prepared bydissolving the test compounds in 10% DMSO and 5% Tween 20 in 85% saline.Test compounds were administered orally (p.o.) by oral gavage. Blood wascollected at selected time points by cardiac puncture under anaesthesiainto heparinized syringes, transferred to microcentrifuge tubes, andcentrifuged at 4500×g for 2 minutes to obtain plasma. Quantitativeanalysis was performed by high performance liquid chromatography tandemmass spectrometry on a triple quadrupole instrument (Agilent 6410) usingmultiple reaction monitoring of selected transitions with olomoucineused as internal standard. Quantitation was performed against a standardcurve ranging from concentrations of 2 nM to 1000 nM in the matrixmeasured. Quality controls were included at the level of 25, 250 and 750nM. If required, samples were diluted in the matrix of interest. Theplasma concentrations of test compounds were measured at 1 hour afteroral dose and expressed relative to a 10 mg/kg dose to provide acomparative estimate of the degree of oral bioavailability.

Biological Data

CHK1 Activity

The following compounds were tested using Assay 1A (Determination ofinhibitor potency vs. CHK1 in DELFIA assay format) or Assay 1B(Determination of inhibitor potency vs. CHK1 in Caliper assay format):

PAPC-A-01, PAPC-A-02, PAPC-A-03, PAPC-A-04, PAPC-A-05, PAPC-A-06,PAPC-A-07, PAPC-A-08, PAPC-A-09, PAPC-A-10, PAPC-B-01, PAPC-B-02,PAPC-B-03, PAPC-B-04, PAPC-B-05, PAPC-B-06, PAPC-B-07, PAPC-B-08,PAPC-B-09, PAPC-B-10, PAPC-B-11.

All of the compounds have a CHK1 IC₅₀ of less than 0.1 μM (100 nM).

The following compounds have a CHK1 IC₅₀ of less than 0.02 μM (20 nM):

PAPC-A-01, PAPC-A-02, PAPC-A-03, PAPC-A-08, PAPC-A-10, PAPC-B-01,PAPC-B-02, PAPC-B-03, PAPC-B-04, PAPC-B-05, PAPC-B-06, PAPC-B-08,PAPC-B-10, PAPC-B-11,

Selectivity for CHK1 vs. CHK2

The following compounds were also tested using Assay 2 (Determination ofinhibitor selectivity for inhibition of CHK1 vs. CHK2):

PAPC-A-01, PAPC-A-02, PAPC-A-03, PAPC-A-07, PAPC-A-08, PAPC-A-09,PAPC-B-02, PAPC-B-03, PAPC-B-04, PAPC-B-06, PAPC-B-10, PAPC-B-11.

All of the compounds have a CHK1 vs. CHK2 selectivity of at least100-fold (i.e., CHK2 IC₅₀/CHK1 IC₅₀>100).

MIA Activity

The following compounds were tested using Assay 3 (Mitosis InhibitionAssay (MIA)):

PAPC-A-01, PAPC-A-02, PAPC-A-03, PAPC-A-04, PAPC-A-05, PAPC-A-06,PAPC-A-07, PAPC-A-08, PAPC-A-09, PAPC-A-10, PAPC-B-01, PAPC-B-02,PAPC-B-03, PAPC-B-04, PAPC-B-05, PAPC-B-06, PAPC-B-07, PAPC-B-08,PAPC-B-09, PAPC-B-10, PAPC-B-11.

The following compounds have a MIA IC₅₀ of less than 0.5 μM:

PAPC-A-01, PAPC-A-02, PAPC-A-03, PAPC-A-04, PAPC-A-05, PAPC-A-06,PAPC-A-07, PAPC-A-08, PAPC-A-09, PAPC-A-10, PAPC-B-01, PAPC-B-02,PAPC-B-03, PAPC-B-04, PAPC-B-05, PAPC-B-06, PAPC-B-08, PAPC-B-10,PAPC-B-11.

Oral Bioavailability

The following compounds were evaluated for oral bioavailability usingthe method described above:

PAPC-A-01, PAPC-A-02, PAPC-A-05, PAPC-A-07, PAPC-A-09, PAPC-B-01,PAPC-B-02, PAPC-B-03, PAPC-B-04, PAPC-B-05, PAPC-B-06, PAPC-B-08,PAPC-B-10.

All of the compounds have oral bioavailability (plasma concentration, 1hour following 10 mg/kg p.o.) of at least 2 nM.

The following compounds have oral bioavailability (plasma concentration,1 hour following 10 mg/kg p.o.) of at least 10 nM.

PAPC-A-01, PAPC-A-02, PAPC-A-05, PAPC-A-07, PAPC-A-09, PAPC-B-02,PAPC-B-05, PAPC-B-06, PAPC-B-08, PAPC-B-10.

The following compounds have oral bioavailability (plasma concentration,1 hour following 10 mg/kg p.o.) of at least 100 nM.

PAPC-A-01, PAPC-A-02, PAPC-A-05, PAPC-A-07, PAPC-B-02, PAPC-B-05:

The following compounds have oral bioavailability (plasma concentration,1 hour following 10 mg/kg p.o.) of at least 500 nM.

PAPC-A-01, PAPC-A-02, PAPC-A-05.

Data for two especially preferred compounds are summarized below:

Compound PAPC-A-01 PAPC-A-02 Structure

CHK1 vs. 0.006 0.008 (μM) CHK1 vs 540-fold >1250-fold CHK2 selectivityMIA IC₅₀ 0.0273 0.029 (μM) Oral bioavail. 555 1214 (plasmaconcentration, 1 hour following 10 mg/kg p.o.) (nM)

In Vivo Study 1: PAPC-a-01 in Transgenic MYCN-Driven Neuroblastoma Model

Spontaneous neuroblastomas arising in hemizygous mice transgenic forTH-MYCN (human MYCN under the control of the rat tyrosine hydroxylasepromoter) were detected by abdominal palpation. Sequential animals withwell-established tumours (30-70 days of age) were randomised to receiveeither test compound (PAPC-A-01) or vehicle until 8-9 mice per groupwere accrued. The test compound (PAPC-A-01) was administered as a singleagent at 100 mg/kg p.o. daily for 7 consecutive days by oral gavage (100μL/10 g body weight) and controls received an equivalent volume ofvehicle (10% DMSO, 5% Tween 20, 85% saline). Neuroblastoma size wasassessed by MRI and by the mass of tumours dissected from the abdominalcavity at the end of therapy.

¹H MRI was performed on a 7T Bruker horizontal bore microimaging system(Bruker Instruments, Ettlingen, Germany) using a 3 cm birdcage coil.Anaesthesia was induced a combination of fentanyl citrate (0.315 mg/mL)plus fluanisone (10 mg/mL) (Hypnorm, Janssen Pharmaceutical, Oxford, UK)and midazolam (5 mg/mL) (Roche, Welwyn Garden City, UK) and water(1:1:2). Animal body temperature was maintained by a warm air blowerthrough the magnet bore.

Anatomical T₂-weighted coronal and transverse images were acquired fromtwenty contiguous 1 mm-thick coronal slices through the mouse abdomen,using a rapid acquisition with refocused echoes (RARE) sequence with 4averages of 128 phase encoding steps over a 3×3 cm field of view, twoechoes of 36 and 132 ms, a TR of 4.5 s and a RARE factor of 8. FollowingMRI, the mice were left to recover on a heat mat for 24 hours. Tumourvolumes were measured using segmentation from regions of interest drawnon every slice from the coronal T₂ weighted images containing tumourusing in-house software (ImageView, working under IDL, ITT, Boulder,Colo., USA). MR imaging in individual mice, pre-treatment and on day 7,showed tumour regression over the 7 day treatment period. See FIG. 1 andFIG. 2.

The mean weight of tumours in control mice at the end of the study was2.1±0.7 g (mean±SD) and in the treated group was 0.3±0.2 g, resulting ina T/C of 13.4%.

In Vivo Study 2: PAPC-a-01 in Combination with Gemcitabine in HT29 HumanColon Carcinoma Xenograft

Female athymic CrTac:NCr-Foxn1nu mice (6-8 weeks old) (Charles River UKLtd., Margate, UK) were kept in a controlled environment in maximisercages with sterile bedding, food and water available ad libitum. Animalsweighed 20.3±2.0 g at the start of the study. Handing and allexperimental procedures were performed under sterile conditions inlaminar flow hoods in accordance with UK Home Office, national and localethical guidelines.

HT29 colon carcinoma cells from the American Type Culture Collection(ATCC, LGC Promochem, Middesex, UK) were harvested from tissue cultureflasks and injected subcutaneously in the right flanks of mice (3million cells per site). Once tumours were established (mean diameter0.55±0.05 cm) mice were randomised to treatment groups (n=6) to receive(a) test compound (PAPC-A-01) (75 mg/kg), (b) gemcitabine (100 mg/kg),(c) a combination (PAPC-A-01) (75 mg/kg) and gemcitabine (100 mg/kg), or(d) relevant vehicles.

Clinical grade gemcitabine hydrochloride (GEMZAR, Eli Lilly, Newmarket,UK) was reconstituted in sterile 0.9% sodium chloride and aliquotsfrozen at −20° C. Gemcitabine was administered intravenously once weeklyvia a lateral tail vein on days 0 (day 5 after tumour cellimplantation), 7 and 14. Test compound (PAPC-A-01) was dissolved in 10%DMSO and diluted out in 5% Tween 20, 85% saline. Test compound(PAPC-A-01) was administered by oral gavage on days 1, 2, 8, 9, 15 and16. The control animals received both vehicles by the appropriate routeon the designated days. The compounds were administered in a volume of100 μL per 10 g body weight.

Animals were observed daily and body weights and tumours measured threetimes weekly. Two perpendicular tumour diameters were used to calculatevolumes using the formula: V=4/3π[(d1+d2)/4]³.

T/C values (volumes of treated tumours vs. controls) were calculatedwith respect to vehicle control tumours or those treated withgemcitabine alone, expressed as a percentage.

On day 24 after the start of therapy, the results were as follows:

Mean tumour volume T/C vs. T/C vs. Group (cm³, mean ± SEM) vehiclecontrols gemcitabine Vehicle controls 1.099 ± 0.304 — — Gemcitabine0.888 ± 0.188 80.8 — PAPC-A-01 1.114 ± 0.257 101.4 — Combination 0.406 ±0.066 36.9 45.7

Gemcitabine alone, at close to the recommended maximum tolerated dose,inhibited tumour growth by approximately 20%. Test compound (PAPC-A-01)alone gave no growth inhibition. The combination therapy of gemcitabinewith test compound (PAPC-A-01) inhibited tumour growth by 63% relativeto controls, and by 54% relative to gemcitabine alone.

The foregoing has described the principles, preferred embodiments, andmodes of operation of the present invention. However, the inventionshould not be construed as limited to the particular embodimentsdiscussed. Instead, the above-described embodiments should be regardedas illustrative rather than restrictive, and it should be appreciatedthat variations may be made in those embodiments by workers skilled inthe art without departing from the scope of the present invention.

REFERENCES

A number of publications are cited herein in order to more fullydescribe and disclose the invention and the state of the art to whichthe invention pertains. Full citations for these references are providedbelow. Each of these references is incorporated herein by reference inits entirety into the present disclosure, to the same extent as if eachindividual reference was specifically and individually indicated to beincorporated by reference.

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1-104. (canceled)
 105. A compound of the following formula, or apharmaceutically acceptable salt thereof:

wherein: —R^(B3) is independently:

each —R^(B3A) is independently —H or saturated aliphatic C₁₋₃alkyl;—R^(A4) is independently —NHR^(A4A), —NR^(A4A) ₂, or —OR^(A4A); each—R^(A4A) is independently saturated aliphatic C₁₋₃alkyl; —R^(A5) isindependently —R^(A5A), —R^(A5B), —R^(A5C), —R^(A5D), R^(A5E), or—R^(A5F); —R^(A5A) is independently:

—R^(A5AA) is saturated aliphatic C₁₋₃alkyl; —R^(A5B) is —CF₃; —R^(A5C)is independently —F, —Cl, —Br, or —I; —R^(A5D) is independently —C≡CH,—C≡C—R^(A5DA), or —C≡C—R^(A5DB)—OH; —R^(A5DA) is saturated aliphaticC₁₋₄alkyl; —R^(A5DB) is saturated aliphatic C₁₋₄alkylene; —R^(A5E) isindependently saturated C₃₋₆cycloalkyl; —R^(A5F) is —C(═O)O—R^(A5FA);and —R^(A5FA) is saturated aliphatic C₁₋₃alkyl.
 106. A compoundaccording to claim 105, wherein —R^(B3) is:


107. A compound according to claim 105, wherein —R^(B3) is:


108. A compound according to claim 105, wherein —R^(B3A) is —H or -Me.109. A compound according to claim 106, wherein —R^(B3A) is -Me.
 110. Acompound according to claim 107, wherein —R^(B3A) is —H or -Me.
 111. Acompound according to claim 108, wherein —R^(A4) is independently—NHR^(A4A) or —NR^(A4A) ₂; wherein each —R^(A4A) is independently -Me or-Et.
 112. A compound according to claim 109, wherein —R^(A4) isindependently —NHR^(A4A) or —NR^(A4A) ₂; wherein each —R^(A4A) isindependently -Me or -Et.
 113. A compound according to claim 110,wherein —R^(A4) is independently —NHR^(A4A) or —NR^(A4A) ₂; wherein each—R^(A4A) is independently -Me or -Et.
 114. A compound according to claim108, wherein —R^(A4) is —OR^(A4A); wherein each —R^(A4A) isindependently -Me or -Et.
 115. A compound according to claim 109,wherein —R^(A4) is —OR^(A4A); wherein each —R^(A4A) is independently -Meor -Et.
 116. A compound according to claim 110, wherein —R^(A4) is—OR^(A4A); wherein each —R^(A4A) is independently -Me or -Et.
 117. Acompound according to claim 105, wherein —R^(A5) is —R^(A5A).
 118. Acompound according to claim 106, wherein —R^(A5) is —R^(A5A).
 119. Acompound according to claim 107, wherein —R^(A5) is —R^(A5A).
 120. Acompound according to claim 108, wherein —R^(A5) is —R^(A5A).
 121. Acompound according to claim 109, wherein —R^(A5) is —R^(A5A).
 122. Acompound according to claim 110, wherein —R^(A5) is —R^(A5A).
 123. Acompound according to claim 111 wherein —R^(A5) is —R^(A5A).
 124. Acompound according to claim 112, wherein —R^(A5) is —R^(A5A).
 125. Acompound according to claim 113, wherein —R^(A5) is —R^(A5A).
 126. Acompound according to claim 114, wherein —R^(A5) is —R^(A5A).
 127. Acompound according to claim 115, wherein —R^(A5) is —R^(A5A).
 128. Acompound according to claim 116, wherein —R^(A5) is —R^(A5A).
 129. Acompound according to claim 105, which is a compound of the followingformula, or a pharmaceutically acceptable salt thereof:


130. A compound according to claim 105, which is a compound of thefollowing formula, or a pharmaceutically acceptable salt thereof:


131. A compound according to claim 105, which is a compound of thefollowing formula, or a pharmaceutically acceptable salt thereof:


132. A compound according to claim 105, which is a compound of one ofthe following formulae, or a pharmaceutically acceptable salt thereof:


133. A compound according to claim 105, which is a compound of thefollowing formula, or a pharmaceutically acceptable salt thereof:


134. A compound according to claim 105, which is a compound of thefollowing formula, or a pharmaceutically acceptable salt thereof:


135. A compound according to claim 105, which is a compound of one ofthe following formulae, or a pharmaceutically acceptable salt thereof:


136. A compound according to claim 105, which is a compound of one ofthe following formulae, or a pharmaceutically acceptable salt thereof:


137. A compound according to claim 105, which is a compound of one ofthe following formulae, or a pharmaceutically acceptable salt thereof:


138. A compound according to claim 105, which is a compound of one ofthe following formulae, or a pharmaceutically acceptable salt, hydrate,or solvate thereof:


139. A compound according to claim 105, which is a compound one of thefollowing formulae, or a pharmaceutically acceptable salt thereof:


140. A compound of the following formula, or a pharmaceuticallyacceptable salt thereof:


141. A pharmaceutical composition comprising a compound according toclaim 105, and a pharmaceutically acceptable carrier or diluent.
 142. Amethod of preparing a pharmaceutical composition comprising the step ofadmixing a compound according to claim 105, and a pharmaceuticallyacceptable carrier or diluent.
 143. A method of treatment of cancercomprising administering to a subject in need of treatment atherapeutically-effective amount of a compound according to claim 105.